Next Article in Journal
In Vitro Activities and Inoculum Effects of Ceftazidime-Avibactam and Aztreonam-Avibactam against Carbapenem-Resistant Enterobacterales Isolates from South Korea
Next Article in Special Issue
Bee Bread Exhibits Higher Antimicrobial Potential Compared to Bee Pollen
Previous Article in Journal
Actinobacillus pleuropneumoniae Eradication with Enrofloxacin May Lead to Dissemination and Long-Term Persistence of Quinolone Resistant Escherichia coli in Pig Herds
Previous Article in Special Issue
The Antibacterial Potential of Honeydew Honey Produced by Stingless Bee (Heterotrigona itama) against Antibiotic Resistant Bacteria
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Antibiotics
Volume 9
Issue 12
10.3390/antibiotics9120911
antibiotics-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Antibiofilm Activity of Heather and Manuka Honeys and Antivirulence Potential of Some of Their Constituents on the DsbA1 Enzyme of Pseudomonas aeruginosa

by
Oscar Shirlaw
1,†,
Zara Billah
1,†,
Baraa Attar
1,
Lisa Hughes
1,
Rana M. Qasaymeh
1,
Veronique Seidel
1,* and
Georgios Efthimiou
2,*
1
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, UK
2
Department of Biomedical and Forensic Sciences, Hardy Building, University of Hull, Hull HU6 7RX, UK
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Antibiotics 2020, 9(12), 911; https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics9120911
Submission received: 29 September 2020 / Revised: 1 December 2020 / Accepted: 10 December 2020 / Published: 15 December 2020
(This article belongs to the Special Issue Honey Bee Products as an Alternative or Complement to Classical Antibiotics)
Browse Figures
Versions Notes

Abstract

:
Heather honey was tested for its effect on the formation of biofilms by Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Salmonella Enteriditis and Acinetobacter baumanii in comparison with Manuka honey. At 0.25 mg/mL, Heather honey inhibited biofilm formation in S. aureus, A. baumanii, E. coli, S. Enteriditis and P. aeruginosa, but promoted the growth of E. faecalis and K. pneumoniae biofilms. Manuka honey inhibited biofilm formation in K. pneumoniae, E. faecalis, and S. Enteriditis, A. baumanii, E. coli and P. aeruginosa, but promoted S. aureus biofilm formation. Molecular docking with Autodock Vina was performed to calculate the predictive binding affinities and ligand efficiencies of Manuka and Heather honey constituents for PaDsbA1, the main enzyme controlling the correct folding of virulence proteins in Pseudomonas aeruginosa. A number of constituents, including benzoic acid and methylglyoxal, present in Heather and/or Manuka honey, revealed high ligand efficiencies for the target enzyme. This helps support, to some extent, the decrease in P. aeruginosa biofilm formation observed for such honeys.

1. Introduction

Antimicrobial drug resistance, particularly in Gram-negative bacteria, is an ever-increasing challenge for healthcare systems worldwide [1]. Alternative treatment options to conventional antibiotics are urgently needed to tackle this global threat [2,3]. This includes the discovery of molecules that could disrupt the ability of pathogens to produce virulence factors [4,5]. In Gram-negative bacteria, various virulence factors are produced under the control of a master virulence regulatory oxidoreductase enzyme called DsbA. The latter catalyses the formation of disulfide bonds in proteins and, in doing so, is instrumental to the process of correct protein folding of bacterial virulence proteins, including type-IV fimbriae, flagellae and adhesion factors that play a central role in biofilm formation [6,7,8,9,10,11]. The disulfide bond forming pathways in Gram-positive bacteria are less well established [12].
Biofilms are formed when microbial communities, held together by a polymeric matrix, attach to surfaces. The formation of microbial biofilms poses significant risks in healthcare settings when pathogens attach to wounds, surfaces, and medical devices [13,14]. The opportunistic nosocomial pathogen, Pseudomonas aeruginosa, is one of the most common bacteria isolated from chronic wounds and has become difficult to eradicate due to its ability to form biofilms [15,16,17,18]. Its DsbA enzyme is an attractive target in the search for new antivirulence agents [19,20].
Products from the beehive have a long history of use as traditional remedies [21], and honey has emerged as a promising topical antibacterial agent [22,23,24,25,26]. Honey is known to contain a diversity of chemicals which vary depending on nectar sources in different geographical locations, harvesting seasons, types of bees foraging and storage of the final product [27,28,29]. The antimicrobial activity of most honeys has been attributed to a high sugar content, low pH, and the ability to produce hydrogen peroxide [22,30]. Other contributors to this activity include plant- and bee-derived chemicals [30]. Both Manuka honey (derived from the nectar of Leptospermum spp.) and Heather honey (from Erica spp.) have a similar pH, low hydrogen peroxide activity and a high sugar content [31] and contain various phytochemicals such as phenolic acids and flavonoids [32,33,34,35]. Manuka honey is very rich in methylglyoxal (MGO), a plant-derived compound formed during storage and used for ‘Unique Manuka Factor’ (UMF) grading [28,29,30,35,36]. Heather honey, on the other hand, is rich in abscisic acid [31,37].
Both honeys have demonstrated antibacterial activity [36,38,39] and an inhibitory effect on polymicrobial biofilms [40]. Manuka honey can disrupt biofilm formation in several pathogens [41,42,43,44,45,46,47,48,49]. To the best of our knowledge, the effect of Heather honey on monobacterial biofilms has never been reported, and neither has the potential affinity of Manuka nor Heather honey constituents for DsbA been predicted. Here, we compared the effect of Heather honey with that of Manuka honey on the formation of biofilms in seven bacteria. We also employed a molecular docking approach to predict the binding affinity of constituents from both honeys towards the P. aeruginosa DsbA enzyme (PaDsbA1).

2. Results

2.1. Determination of the Time Required for Optimal Biofilm Formation

Optimal biofilm formation (OD550 nm 0.8–1.7) by all bacterial species was obtained after a 24 h incubation period. This time point was selected to further study biofilm formation in subsequent experiments. High amounts of biofilm were also observed after 48 h (OD550 nm 0.7–1.6), less so after 72 and 96 h (Supplementary Materials Figure S1).

2.2. Antibiofilm Activity

At 0.25 mg/mL, Manuka honey showed the strongest inhibition of biofilm formation in K. pneumoniae, E. faecalis, and S. Enteriditis (92.8, 78.0, and 65.7% inhibition, respectively). It also inhibited biofilm formation in A. baumanii, E. coli and P. aeruginosa. Unlike Manuka honey, which increased S. aureus biofilm formation, Heather honey inhibited biofilm formation in S. aureus (69.6%). It decreased biofilm formation in A. baumanii, E. coli, S. Enteriditis and P. aeruginosa, but promoted the growth of E. faecalis biofilms (Figure 1). Oleanolic acid decreased biofilm formation in all bacteria (≥50% in all cases except for E. coli) (Figure 2).

2.3. Molecular Docking of Honey Constituents against P. aeruginosa DsbA1

A guided docking approach was used to predict the binding affinities and ligand efficiency indices of 56 constituents of Manuka and Heather honey towards PaDsbA1 (Supplementary Materials Table S1). The best ligand efficiencies were obtained for benzoic acid (0.60), 5-methyl-3-furancarboxylic acid (0.57), methylglyoxal (0.56) and 5-hydroxymethyl-2-furaldehyde (0.56) (Table 1). The molecular interactions of each of these ligands with PaDsbA1 are detailed in Table 1 and in Figure 3, Figure 4, Figure 5 and Figure 6. Benzoic acid strongly interacted with PaDsbA1 via two hydrogen bonds (contact distances < 2.5 Å) to Tyr148 and Pro20 and hydrophobic bonds with Leu144, Leu63, Val30, and Val61.

3. Discussion

Heather honey has been the subject of a limited number of studies investigating its effect on bacterial biofilms. Only one study to date has reported its inhibitory activity on mixed Candida–Pseudomonas biofilms [40]. This prompted us to test its activity against a range of Gram-positive and Gram-negative single-species biofilms. This was done using a crystal violet assay, a commonly-used indirect method of biofilm quantification [50], and in comparison with Manuka honey, already known for its ability to decrease bacterial biofilm formation [41,42,43,44,45,46,47,48,49]. The decrease in S. aureus, A. baumanii, E. coli, S. Enteriditis and P. aeruginosa biofilm formation observed for Heather honey is reported here for the first time. Manuka honey decreased biofilm formation in P. aeruginosa, E. faecalis, A. baumanii, and E. coli, in agreement with previous studies [45,46,47,48,49]. Its effect on K. pneumoniae and S. Enteriditis biofilms has not been previously reported. Heather and Manuka honey, both tested at sub-inhibitory concentrations, increased biofilm formation in the Gram-positive bacteria E. faecalis and S. aureus, respectively. Such an effect has been observed in other investigations [29,51]. It has been suggested that the decrease in biofilm formation reported for Manuka honey could be linked to the presence of phenolic compounds, bee defensin-1 and/or MGO. The latter is able to inhibit biofilm formation by altering the structures of bacterial fimbriae and flagellae [29,36]. Oleanolic acid is known to decrease biofilm formation [52,53,54,55], but this is the first report of such an effect against E. faecalis, S. aureus, E. coli, K. pneumoniae, A. baumanii and S. Enteriditis biofilms.
The DsbA enzymes of Gram-negative bacteria have emerged as attractive targets for the discovery and development of new antivirulence agents including from natural sources [56,57]. There are two main structural classes of DsbA enzymes, DsbA-I and DsbA-II. Both classes contain proteins that share highly conserved residues in their catalytic active sites, and also several identical hydrophobic amino acids adjacent to this site. DsbA-I proteins have a unique groove on their non-catalytic face, opposite to the active site surface. PaDsbA1 was identified as the main DsbA-I type enzyme in P. aeruginosa [20]. Our molecular docking study was conducted to predict the binding affinity of honey constituents for the groove on the non-catalytic face the PaDsbA1 [11,20].
Our predictive in silico analysis revealed that benzoic acid, a constituent of both Heather [58] and Manuka honey [59], could bind with a high efficiency into the pocket of PaDsbA1 where the control ligand fits [20]. This binding occurs via H-bond interactions between the hydroxyl hydrogen of benzoic acid and the carbonyl oxygen of Pro20, and between the hydroxyl oxygen of benzoic acid and the phenolic hydrogen of Tyr148. Interestingly, previous studies reported that benzoic acid could attenuate the virulence of P. aeruginosa in plants and nematodes through inhibiting the production of virulence factors such as pyocyanin, and reducing total protease and elastase activity [60]. The latter exoenzyme plays a critical role in the virulence of P. aeruginosa and requires PaDsbA1 for its biogenesis [6,10,20]. In the case of Manuka honey, our predictions also revealed that MGO had a high binding efficiency for PaDsbA1. This could support the decrease in P. aeruginosa biofilm formation for this type of honey via alteration of fimbriae and flagellae. Further work is required to confirm this hypothesis and test MGO and other honey constituents showing high predictive binding efficiencies in our in silico screening for their ability to prevent biofilms in wild type bacteria and ΔDsbA mutants.

4. Materials and Methods

4.1. Bacterial Strains, Culture Conditions, and Inoculum Preparation

Staphylococcus aureus (ATCC 43300), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603), Enterococcus faecalis (ATCC 51299) and Acinetobacter baumanii (ATCC 19606) were obtained from the American Type Culture Collection. Salmonella enteriditis (NCTC 4444) was obtained from the National Collection of Type Cultures. In preparation for antibiofilm activity screening, each strain was grown in tryptone soya broth (TSB; Oxoid, UK) at 37 °C for 4 h (late exponential phase) under continuous shaking (250 rpm).

4.2. Honey Sources and Preparation of Samples

Heather honey was kindly provided by the Scottish Bee Company. Manuka honey (UMF 10+) was obtained from Wilkin & Sons Ltd., Tiptree, UK. An exactly weighed amount (around 1 g) of each sample was dissolved in water and then filter-sterilised (0.22 μm disks, Sartorius, UK) to afford stock solutions of Heather honey and Manuka honey (100 mg/mL).

4.3. Antibiofilm Assay

This was performed according to a previous method with some modifications [61]. Briefly, 100 μL of each bacterial inoculum (2.5 × 105 CFU/mL) was added to the wells of a 24-flat well polystyrene plate, containing TSB (1.8 mL). Heather and Manuka honeys were tested at 0.25 mg/mL. Wells containing TSB only were used as the sterile controls. Oleanolic acid (Sigma-Aldrich, Gillingham, UK), a plant-derived triterpenoid which inhibits the formation of biofilms in Gram-positive and Gram-negative bacteria, was used as a positive control [52,53,54,55]. Following inoculation, the plates were incubated without shaking at 37 °C for 24, 48, 72, and 96 h to allow the formation of a biofilm at the bottom of the wells as well as to determine the time point at which the maximum formation of biofilm occurred. After this period, the supernatants were removed, and the biofilms were washed (×3) with sterile distilled water. The biofilms were then stained with 1% crystal violet for 5 min, and further washed (×3) with tap water. A de-staining step was performed using a 7:3 (v/v) mixture of ethanol and acetone and the OD of the suspension was measured at 550 nm. The percentage of biofilm inhibition was calculated as: (ODcontrol − ODsample) × 100/ODcontrol.

4.4. Molecular Docking Experiment

4.4.1. Protein Preparation

BIOVIA Discovery Studio Visualizer v.4.5 (Accelrys) was used to remove all water molecules and hetero-atoms from the three-dimensional crystal structure of the PaDsbA1 protein (PDB ID:5DCH) which was retrieved, complexed with its ligand inhibitor (MIPS-0000851), from the RCSB Protein Data Bank (http://www.pdb.org). AutoDock Tools v. 1.5.6rc3 was subsequently used to prepare a PDBQT file of the target protein containing added polar hydrogen atoms [62].

4.4.2. Ligand Preparation

Flavonoids, phenolic acids, and other miscellaneous compounds previously reported in Manuka and/or Heather honeys [35,37,58,59,63,64,65,66,67,68,69,70,71,72,73,74] were selected as ligands for the docking experiment. All chemical structures were retrieved from SciFinder (https://scifinder.cas.org/scifinder/login). Each structure was exported to ChemOffice v.16.0 and geometry-optimised using MM2 energy minimization [75]. The structure of the ligand inhibitor (MIPS-0000851) was obtained from the RCSB Protein Data Bank (http://www.pdb.org). Docking files for all ligands were prepared using AutoDock Tools v. 1.5.6rc3 [62]. Rigid docking was performed to minimise standard errors likely due to ligands with many active rotatable bonds [76]. Gasteiger charges were assigned [77] and the files were saved as PDBQT formats in preparation for docking.

4.4.3. Grid Box Preparation and Docking Studies

The size of the searching space around the PaDsbA1 binding site residues was defined with grid box parameters prepared using AutoDock Tools v. 1.5.6rc3, and the molecular docking was done with AutoDock Vina v. 1.1.2 [76]. The centre of the grid box was set to x = 21.7657, y = 35.6275, z = −2.0723. Its size was 22 × 22 × 22 points in the x, y, z dimensions. The spacing was set at 1 Å. MIPS-0000851, a known PaDsbA1 inhibitor, was retrieved from its co-crystallised complex with the target protein and re-docked as a control against the enzyme to validate the docking conditions. Different orientations of the ligands were searched and ranked based on their energy scores. Upon visual inspection of all binding poses obtained, only poses with the lowest root mean square deviation (RMSD) value (threshold < 1.00 Å) were considered to provide a high accuracy of docking. The default values set in Autodock Vina were used as the parameters for the rigid-ligand docking (exhaustiveness = 8). The docking scores were calculated as the predicted free energies of binding (ΔG in kcal/mol). Ligand efficiencies were calculated as the ratio of ΔG to the number of heavy atoms (NHA) for each ligand (LE = –(ΔG)/NHA) [78] (Table 1 and Table S1).

4.4.4. Protein-Ligand Interactions and Predictive Inhibition

BIOVIA Discovery Studio Visualizer v.4.5 (Accelrys) was used to identify the H-bonds and non-bonding interactions between the ligand docking poses and the binding site of PaDsbA1 (Table 1 and Supplementary Materials Table S1).

5. Conclusions

We have shown that benzoic acid (in Manuka and Heather honey) and other small molecules including MGO (in Manuka honey) have the potential to target virulence in P. aeruginosa. Further investigations should aim to analyse the effect of Heather honey on P. aeruginosa biofilm morphology using confocal laser scanning microscopy and identify the nature of its active constituent(s). Additional molecular docking could be performed to establish whether binding of honey constituents occurs in other DsbA enzymes that share a similarity with PaDsbA1 (e.g., Acinetobacter baumannii AbDsbA). As Manuka and Heather honeys display bactericidal activity against P. aeruginosa [79], they represent an interesting alternative/complementary option to treat persistent wounds infected with this pathogen.

Supplementary Materials

The following are available online at https://0-www-mdpi-com.brum.beds.ac.uk/2079-6382/9/12/911/s1, Figure S1: Time course assay for the determination of the optimal bacterial biofilm formation time, Table S1: Docking scores and ligand efficiencies of Manuka and/or Heather honey constituents towards PaDsbA1.

Author Contributions

Conceptualization, V.S. and G.E.; methodology, V.S. and G.E.; software, V.S.; validation, V.S. and G.E.; formal analysis, V.S. and G.E.; investigation, O.S., R.M.Q., Z.B., L.H., B.A., V.S. and G.E.; resources, V.S. and G.E.; data curation, V.S. and G.E.; writing—original draft preparation, V.S. and G.E.; writing—review and editing, V.S. and G.E.; visualization, V.S. and G.E.; supervision, V.S. and G.E.; project administration, V.S. and G.E. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Acknowledgments

We are grateful to Iain and Suzie Millar from the Scottish Bee Company for providing us with Heather honey.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Arzanlou, M.; Chai, W.C.; Venter, H. Intrinsic, adaptive and acquired antimicrobial resistance in Gram-negative bacteria. Essays Biochem. 2017, 61, 49–59. [Google Scholar] [CrossRef]
  2. Rossiter, S.E.; Fletcher, M.H.; Wuest, W.M. Natural Products as Platforms to Overcome Antibiotic Resistance. Chem. Rev. 2017, 117, 12415–12474. [Google Scholar] [CrossRef] [PubMed]
  3. Chandra, H.; Bishnoi, P.; Yadav, A.; Patni, B.; Mishra, A.P.; Nautiyal, A.R. Antimicrobial Resistance and the Alternative Resources with Special Emphasis on Plant-Based Antimicrobials—A Review. Plants 2017, 6, 16. [Google Scholar] [CrossRef] [PubMed]
  4. Dickey, S.W.; Cheung, G.Y.C.; Otto, M. Different drugs for bad bugs: Antivirulence strategies in the age of antibiotic resistance. Nat. Rev. Drug Discov. 2017, 16, 457–471. [Google Scholar] [CrossRef]
  5. Heras, B.; Scanlon, M.J.; Martin, J.L. Targeting virulence not viability in the search for future antibacterials. Br. J. Clin. Pharmacol. 2015, 79, 208–215. [Google Scholar] [CrossRef] [PubMed]
  6. Shouldice, S.R.; Heras, B.; Walden, P.M.; Totsika, M.; Schembri, M.A.; Martin, J.L. Structure and Function of DsbA, a Key Bacterial Oxidative Folding Catalyst. Antioxidants Redox Signal. 2011, 14, 1729–1760. [Google Scholar] [CrossRef] [Green Version]
  7. Yu, J. DsbA: A protein-folding catalyst contributing to bacterial virulence. Microbes Infect. 1999, 1, 1221–1228. [Google Scholar] [CrossRef]
  8. Lee, Y.; Kim, Y.; Yeom, S.; Kim, S.; Park, S.; Jeon, C.O.; Park, W. The role of disulfide bond isomerase A (DsbA) of Escherichia coli O157:H7 in biofilm formation and virulence. FEMS Microbiol. Lett. 2008, 278, 213–222. [Google Scholar] [CrossRef] [Green Version]
  9. Bocian-Ostrzycka, K.M.; Grzeszczuk, M.J.; Banaś, A.M.; Jagusztyn-Krynicka, E.K. Bacterial thiol oxidoreductases—From basic research to new antibacterial strategies. Appl. Microbiol. Biotechnol. 2017, 101, 3977–3989. [Google Scholar] [CrossRef] [Green Version]
  10. Lasica, A.M.; Jagusztyn-Krynicka, E.K. The role of Dsb proteins of Gram-negative bacteria in the process of pathogenesis. FEMS Microbiol. Rev. 2007, 31, 626–636. [Google Scholar] [CrossRef] [Green Version]
  11. Totsika, M.; Vagenas, D.; Paxman, J.J.; Wang, G.; Dhouib, R.; Sharma, P.; Martin, J.L.; Scanlon, M.J.; Heras, B. Inhibition of Diverse DsbA Enzymes in Multi-DsbA Encoding Pathogens. Antioxid. Redox Signal. 2018, 29, 653–666. [Google Scholar] [CrossRef] [PubMed]
  12. Reardon-Robinson, M.E.; Ton-That, H. Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria. J. Bacteriol. 2015, 198, 746–754. [Google Scholar] [CrossRef] [Green Version]
  13. Kumar, A.; Alam, A.; Rani, M.; Ehtesham, N.Z.; Hasnain, S.E. Biofilms: Survival and defense strategy for pathogens. Int. J. Med. Microbiol. 2017, 307, 481–489. [Google Scholar] [CrossRef]
  14. Muhammad, M.H.; Idris, A.L.; Fan, X.; Guo, Y.; Yu, Y.; Jin, X.; Qiu, J.; Guan, X.; Huang, T. Beyond Risk: Bacterial Biofilms and Their Regulating Approaches. Front. Microbiol. 2020, 11, 928. [Google Scholar] [CrossRef]
  15. Lee, K.; Yoon, S.S. Pseudomonas aeruginosa Biofilm, a Programmed Bacterial Life for Fitness. J. Microbiol. Biotechnol. 2017, 27, 1053–1064. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Pang, Z.; Raudonis, R.; Glick, B.R.; Lin, T.-J.; Cheng, Z. Antibiotic resistance in Pseudomonas aeruginosa: Mechanisms and alternative therapeutic strategies. Biotechnol. Adv. 2019, 37, 177–192. [Google Scholar] [CrossRef] [PubMed]
  17. Clinton, A.; Carter, T. Chronic Wound Biofilms: Pathogenesis and Potential Therapies. Lab. Med. 2015, 46, 277–284. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Serra, R.; Grande, R.; Butrico, L.; Rossi, A.; Settimio, U.F.; Caroleo, B.; Amato, B.; Gallelli, L.; De Franciscis, S. Chronic wound infections: The role of Pseudomonas aeruginosa and Staphylococcus aureus. Expert Rev. Anti-Infect. Ther. 2015, 13, 605–613. [Google Scholar] [CrossRef]
  19. Ha, U.-H.; Wang, Y.; Jin, S. DsbA of Pseudomonas aeruginosa Is Essential for Multiple Virulence Factors. Infect. Immun. 2003, 71, 1590–1595. [Google Scholar] [CrossRef] [Green Version]
  20. Mohanty, B.; Rimmer, K.; McMahon, R.M.; Headey, S.J.; Vazirani, M.; Shouldice, S.R.; Coinçon, M.; Tay, S.; Morton, C.J.; Simpson, J.S.; et al. Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1. PLoS ONE 2017, 12, e0173436. [Google Scholar] [CrossRef] [Green Version]
  21. Viuda-Martos, M.; Ruiz-Navajas, Y.; Fernández-López, J.; Pérez-Álvarez, J. Functional Properties of Honey, Propolis, and Royal Jelly. J. Food Sci. 2008, 73, R117–R124. [Google Scholar] [CrossRef] [PubMed]
  22. Mandal, M.D.; Mandal, S. Honey: Its medicinal property and antibacterial activity. Asian Pac. J. Trop. Biomed. 2011, 1, 154–160. [Google Scholar] [CrossRef] [Green Version]
  23. Hussain, M.B. Role of Honey in Topical and Systemic Bacterial Infections. J. Altern. Complement. Med. 2018, 24, 15–24. [Google Scholar] [CrossRef]
  24. Temaru, E.; Shimura, S.; Amano, K.; Karasawa, T. Antibacterial activity of honey from stingless honeybees (Hymenoptera; Apidae; Meliponinae). Pol. J. Microbiol. 2007, 56, 281–285. [Google Scholar] [PubMed]
  25. Maddocks, S.E.; Jenkins, R.E. Honey: A sweet solution to the growing problem of antimicrobial resistance? Future Microbiol. 2013, 8, 1419–1429. [Google Scholar] [CrossRef]
  26. Minden-Birkenmaier, B.A.; Bowlin, G.L. Honey-Based Templates in Wound Healing and Tissue Engineering. Bioengineering 2018, 5, 46. [Google Scholar] [CrossRef] [Green Version]
  27. Da Silva, P.M.; Gauche, C.; Gonzaga, L.V.; Costa, A.C.O.; Fett, R. Honey: Chemical composition, stability and authenticity. Food Chem. 2016, 196, 309–323. [Google Scholar] [CrossRef]
  28. Albaridi, N.A. Antibacterial Potency of Honey. Int. J. Microbiol. 2019, 2019, 1–10. [Google Scholar] [CrossRef]
  29. Nolan, V.C.; Harrison, J.; Cox, J. Dissecting the Antimicrobial Composition of Honey. Antibiotics 2019, 8, 251. [Google Scholar] [CrossRef] [Green Version]
  30. Kwakman, P.H.S.; Zaat, S.A.J. Antibacterial components of honey. IUBMB Life 2012, 64, 48–55. [Google Scholar] [CrossRef]
  31. Fyfe, L.; Okoro, P.; Paterson, E.; Coyle, S.; McDougall, G.J. Compositional analysis of Scottish honeys with antimicrobial activity against antibiotic-resistant bacteria reveals novel antimicrobial components. LWT Food Sci. Technol. 2017, 79, 52–59. [Google Scholar] [CrossRef]
  32. Dezmirean, G.I.; Mărghitaş, L.A.; Bobiş, O.; Dezmirean, D.S.; Bonta, V.; Erler, S. Botanical Origin Causes Changes in Nutritional Profile and Antioxidant Activity of Fermented Products Obtained from Honey. J. Agric. Food Chem. 2012, 60, 8028–8035. [Google Scholar] [CrossRef] [PubMed]
  33. Karabagias, I.K.; Maia, M.; Karabagias, V.K.; Gatzias, I.; Badeka, A.V. Characterization of Eucalyptus, Chestnut and Heather Honeys from Portugal Using Multi-Parameter Analysis and Chemo-Calculus. Foods 2018, 7, 194. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Kavanagh, S.; Gunnoo, J.; Passos, T.M.; Stout, J.C.; White, B. Physicochemical properties and phenolic content of honey from different floral origins and from rural versus urban landscapes. Food Chem. 2019, 272, 66–75. [Google Scholar] [CrossRef]
  35. Mavric, E.; Wittmann, S.; Barth, G.; Henle, T. Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand. Mol. Nutr. Food Res. 2008, 52, 483–489. [Google Scholar] [CrossRef]
  36. Johnston, M.; McBride, M.; Dahiya, D.; Owusu-Apenten, R.; Nigam, P.S. Antibacterial activity of Manuka honey and its components: An overview. AIMS Microbiol. 2018, 4, 655–664. [Google Scholar] [CrossRef]
  37. Ferreres, F.; Andrade, P.; Tomás-Barberán, F.A. Natural Occurrence of Abscisic Acid in Heather Honey and Floral Nectar. J. Agric. Food Chem. 1996, 44, 2053–2056. [Google Scholar] [CrossRef]
  38. Salonen, A.; Virjamo, V.; Tammela, P.; Fauch, L.; Julkunen-Tiitto, R. Screening bioactivity and bioactive constituents of Nordic unifloral honeys. Food Chem. 2017, 237, 214–224. [Google Scholar] [CrossRef] [Green Version]
  39. Feás, X.; Iglesias, A.; Rodrigues, S.; Estevinho, L.M. Effect of Erica sp. Honey against Microorganisms of Clinical Importance: Study of the Factors Underlying this Biological Activity. Molecules 2013, 18, 4233–4246. [Google Scholar] [CrossRef] [Green Version]
  40. Fernandes, L.; Oliveira, A.; Henriques, M.; Rodrigues, M.E. Honey as a Strategy to Fight Candida tropicalis in Mixed-Biofilms with Pseudomonas aeruginosa. Antibiotics 2020, 9, 43. [Google Scholar] [CrossRef] [Green Version]
  41. Lu, J.; Turnbull, L.; Burke, C.M.; Liu, M.; Carter, D.A.; Schlothauer, R.C.; Whitchurch, C.B.; Harry, E.J. Manuka-type honeys can eradicate biofilms produced by Staphylococcus aureusstrains with different biofilm-forming abilities. PeerJ 2014, 2, e326. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Paramasivan, S.; Drilling, A.; Jardeleza, C.; Jervis-Bardy, J.; Vreugde, S.; Wormald, P.J. Methylglyoxal-augmented Manuka honey as a topical anti-Staphylococcus aureus biofilm agent: Safety and efficacy in an in vivo model. Int. Forum Allergy Rhinol. 2014, 4, 187–195. [Google Scholar] [CrossRef]
  43. Piotrowski, M.; Karpiński, P.; Pituch, H.; Van Belkum, A.; Obuch-Woszczatyński, P. Antimicrobial effects of Manuka honey on in vitro biofilm formation by Clostridium difficile. Eur. J. Clin. Microbiol. Infect. Dis. 2017, 36, 1661–1664. [Google Scholar] [CrossRef] [PubMed]
  44. Hannan, A.; Bajwa, A.E.; Riaz, S.; Arshad, U.; Saleem, S.; Bajwa, U.I. In vitro Salmonella typhi biofilm formation on gallstones and its disruption by Manuka honey. Pak. J. Pharm. Sci. 2018, 31, 129–135. [Google Scholar] [PubMed]
  45. Maddocks, S.E.; Jenkins, R.E.; Rowlands, R.S.; Purdy, K.J.; Cooper, R.A. Manuka honey inhibits adhesion and invasion of medically important wound bacteriain vitro. Future Microbiol. 2013, 8, 1523–1536. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Roberts, A.E.L.; Powell, L.C.; Pritchard, M.F.; Thomas, D.W.; Jenkins, R.E. Anti-pseudomonad Activity of Manuka Honey and Antibiotics in a Specialized ex vivo Model Simulating Cystic Fibrosis Lung Infection. Front. Microbiol. 2019, 10, 869. [Google Scholar] [CrossRef] [Green Version]
  47. Lu, J.; Cokcetin, N.N.; Burke, C.M.; Turnbull, L.; Liu, M.; Carter, D.; Whitchurch, C.B.; Harry, E.J. Honey can inhibit and eliminate biofilms produced by Pseudomonas aeruginosa. Sci. Rep. 2019, 9, 18160. [Google Scholar] [CrossRef] [Green Version]
  48. Abbas, H.A.; Gad, A.I. Eradication of biofilms formed by bacteria isolated from diabetic foot infections by potential antibiofilm agents alone and in combination with ciprofloxacin. Afr. J. Microbiol. Res. 2014, 8, 3882–3892. [Google Scholar]
  49. Halstead, F.D.; Webber, M.A.; Rauf, M.; Burt, R.; Dryden, M.; Oppenheim, B.A. In vitro activity of an engineered honey, medical-grade honeys, and antimicrobial wound dressings against biofilm-producing clinical bacterial isolates. J. Wound Care 2016, 25, 93–102. [Google Scholar] [CrossRef] [Green Version]
  50. Xu, Z.; Liang, Y.; Lin, S.; Chen, D.; Li, B.; Li, L.; Deng, Y. Crystal Violet and XTT Assays on Staphylococcus aureus Biofilm Quantification. Curr. Microbiol. 2016, 73, 474–482. [Google Scholar] [CrossRef]
  51. Sindi, A.; Chawn, M.V.B.; Hernandez, M.E.; Green, K.; Islam, K.; Locher, C.; Hammer, K. Anti-biofilm effects and characterisation of the hydrogen peroxide activity of a range of Western Australian honeys compared to Manuka and multifloral honeys. Sci. Rep. 2019, 9, 17666. [Google Scholar] [CrossRef] [PubMed]
  52. Zhou, L.; Ding, Y.; Chen, W.; Zhang, P.; Chen, Y.; Lv, X. Thein vitrostudy of ursolic acid and oleanolic acid inhibiting cariogenic microorganisms as well as biofilm. Oral Dis. 2012, 19, 494–500. [Google Scholar] [CrossRef]
  53. Kurek, A.; Markowska, K.; Grudniak, A.M.; Janiszowska, W.; Wolska, K.I. The effect of oleanolic and ursolic acids on the hemolytic properties and biofilm formation of Listeria monocytogenes. Pol. J. Microbiol. 2014, 63, 21–25. [Google Scholar] [CrossRef] [PubMed]
  54. Lawrence, J.A.; Huang, Z.; Rathinavelu, S.; Hu, J.-F.; Garo, E.; Ellis, M.; Norman, V.L.; Buckle, R.; Williams, R.B.; Starks, C.M.; et al. Optimized plant compound with potent anti-biofilm activity across gram-negative species. Bioorg. Med. Chem. 2020, 28, 115229. [Google Scholar] [CrossRef] [PubMed]
  55. Wu, C.D. Grape Products and Oral Health. J. Nutr. 2009, 139, 1818S–1823S. [Google Scholar] [CrossRef] [Green Version]
  56. Smith, R.P.; Paxman, J.; Scanlon, M.J.; Heras, B. Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents. Molecules 2016, 21, 811. [Google Scholar] [CrossRef] [Green Version]
  57. Mirza, Z.R.M.H.; Hasan, T.; Seidel, V.; Yu, J. Geraniol as a novel antivirulence agent against bacillary dysentery-causing Shigella sonnei. Virulence 2018, 9, 450–455. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Machado, A.M.; Miguel, M.G.; Vilas-Boas, M.; Figueiredo, A.C. Honey Volatiles as a Fingerprint for Botanical Origin—A Review on their Occurrence on Monofloral Honeys. Molecules 2020, 25, 374. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  59. Tan, S.T.; Holland, P.T.; Wilkins, A.L.; Molan, P.C. Extractives from New Zealand honeys. 1. White clover, manuka and kanuka unifloral honeys. J. Agric. Food Chem. 1988, 36, 453–460. [Google Scholar] [CrossRef]
  60. Prithiviraj, B.; Bais, H.P.; Weir, T.; Suresh, B.; Najarro, E.H.; Dayakar, B.V.; Schweizer, H.P.; Vivanco, J.M. Down Regulation of Virulence Factors of Pseudomonas aeruginosa by Salicylic Acid Attenuates Its Virulence on Arabidopsis thaliana and Caenorhabditis elegans. Infect. Immun. 2005, 73, 5319–5328. [Google Scholar] [CrossRef] [Green Version]
  61. Woodward, M.J.; Sojka, M.; Sprigings, K.A.; Humphrey, T.J. The role of SEF14 and SEF17 fimbriae in the adherence of Salmonella enterica serotype Enteritidis to inanimate surfaces. J. Med. Microbiol. 2000, 49, 481–487. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30, 2785–2791. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Weston, R.J.; Mitchell, K.R.; Allen, K.L. Antibacterial phenolic components of New Zealand manuka honey. Food Chem. 1999, 64, 295–301. [Google Scholar] [CrossRef]
  64. Yao, L.; Datta, N.; Tomás-Barberán, F.A.; Ferreres, F.; Martos, I.; Singanusong, R. Flavonoids, phenolic acids and abscisic acid in Australian and New Zealand Leptospermum honeys. Food Chem. 2003, 81, 159–168. [Google Scholar] [CrossRef]
  65. Daher, S.; Gülaçar, F.O. Identification of New Aromatic Compounds in the New Zealand Manuka Honey by Gas Chromatography-Mass Spectrometry. E-J. Chem. 2010, 7, 7. [Google Scholar] [CrossRef]
  66. Khalil, M.; Alam, N.; Moniruzzaman, M.; Sulaiman, S.; Gan, S.H. Phenolic Acid Composition and Antioxidant Properties of Malaysian Honeys. J. Food Sci. 2011, 76, C921–C928. [Google Scholar] [CrossRef]
  67. Oelschlaegel, S.; Gruner, M.; Wang, P.-N.; Boettcher, A.; Koelling-Speer, I.; Speer, K. Classification and Characterization of Manuka Honeys Based on Phenolic Compounds and Methylglyoxal. J. Agric. Food Chem. 2012, 60, 7229–7237. [Google Scholar] [CrossRef]
  68. Sergiel, I.; Pohl, P.; Biesaga, M. Characterisation of honeys according to their content of phenolic compounds using high performance liquid chromatography/tandem mass spectrometry. Food Chem. 2014, 145, 404–408. [Google Scholar] [CrossRef]
  69. Rückriemen, J.; Schwarzenbolz, U.; Adam, S.; Henle, T. Identification and Quantitation of 2-Acetyl-1-pyrroline in Manuka Honey (Leptospermum scoparium). J. Agric. Food Chem. 2015, 63, 8488–8492. [Google Scholar] [CrossRef]
  70. Grainger, M.N.; Manley-Harris, M.; Lane, J.R.; Field, R.J. Kinetics of conversion of dihydroxyacetone to methylglyoxal in New Zealand mānuka honey: Part I—Honey systems. Food Chem. 2016, 202, 484–491. [Google Scholar] [CrossRef]
  71. Álvarez-Suarez, J.M.; Giampieri, F.; Cordero, M.; Gasparrini, M.; Forbes-Hernández, T.Y.; Mazzoni, L.; Afrin, S.; Beltrán-Ayala, P.; González-Paramás, A.M.; Santos-Buelga, C.; et al. Activation of AMPK/Nrf2 signalling by Manuka honey protects human dermal fibroblasts against oxidative damage by improving antioxidant response and mitochondrial function promoting wound healing. J. Funct. Foods 2016, 25, 38–49. [Google Scholar] [CrossRef]
  72. Daniels, B.J.; Prijic, G.; Meidinger, S.; Loomes, K.M.; Stephens, J.M.; Schlothauer, R.C.; Furkert, D.P.; Brimble, M.A. Isolation, Structural Elucidation, and Synthesis of Lepteridine from Mānuka (Leptospermum scoparium) Honey. J. Agric. Food Chem. 2016, 64, 5079–5084. [Google Scholar] [CrossRef]
  73. Russell, K.M.; Molan, P.C.; Wilkins, A.L.; Holland, P.T. Identification of some antibacterial constituents of New Zealand manuka honey. J. Agric. Food Chem. 1990, 38, 10–13. [Google Scholar] [CrossRef]
  74. Yin, Y.; Chen, H.; Chen, L.; Bie, X.; Ding, T.; Zhang, X.; Wu, B.; Shen, C.; Zhang, R. Determination of three coriaria lactones in honey by ultra high performance liquid chromatography-high resolution mass spectrometry. Chin. J. Chromatogr. 2015, 33, 711. [Google Scholar] [CrossRef] [PubMed]
  75. Allinger, N.L. ChemInform Abstract: Conformational Analysis. 130. MM2. A Hydrocarbon Force Field Utilizing V1 And V2 Torsional Terms. Chem. Informationsdienst 1978, 9, 130. [Google Scholar] [CrossRef]
  76. Trott, O.; Olson, A.J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2009, 31, 455–461. [Google Scholar] [CrossRef] [Green Version]
  77. Gasteiger, J.; Marsili, M. Iterative partial equalization of orbital electronegativity—A rapid access to atomic charges. Tetrahedron 1980, 36, 3219–3228. [Google Scholar] [CrossRef]
  78. García-Sosa, A.T.; Hetényi, C.; Maran, U. Drug efficiency indices for improvement of molecular docking scoring functions. J. Comput. Chem. 2010, 31, 174–184. [Google Scholar] [CrossRef]
  79. Mullai, V.; Menon, T. Bactericidal Activity of Different Types of Honey against Clinical and Environmental Isolates of Pseudomonas aeruginosa. J. Altern. Complement. Med. 2007, 13, 439–442. [Google Scholar] [CrossRef] [Green Version]
Antibiotics 09 00911 g001 550
Figure 1. Inhibition of bacterial biofilm formation by Heather and Manuka honeys (0.25 mg/mL).
Figure 1. Inhibition of bacterial biofilm formation by Heather and Manuka honeys (0.25 mg/mL).
Antibiotics 09 00911 g001
Antibiotics 09 00911 g002 550
Figure 2. Inhibition of biofilm formation by oleanolic acid (OA) tested at a concentration of 0.0625 mg/mL.
Figure 2. Inhibition of biofilm formation by oleanolic acid (OA) tested at a concentration of 0.0625 mg/mL.
Antibiotics 09 00911 g002
Antibiotics 09 00911 g003 550
Figure 3. (a) Docked pose of benzoic acid in the PaDsbA1 binding site showing molecular interactions—hydrogen and hydrophobic bonds shown as green and pink/purple dashed lines, respectively; (b) 2D plot of interactions between benzoic acid and key residues of PaDsbA1 generated by BIOVIA Discovery Studio visualizer.
Figure 3. (a) Docked pose of benzoic acid in the PaDsbA1 binding site showing molecular interactions—hydrogen and hydrophobic bonds shown as green and pink/purple dashed lines, respectively; (b) 2D plot of interactions between benzoic acid and key residues of PaDsbA1 generated by BIOVIA Discovery Studio visualizer.
Antibiotics 09 00911 g003
Antibiotics 09 00911 g004 550
Figure 4. (a) Docked pose of 5-methyl-3-furancarboxylic acid in the PaDsbA1 binding site showing molecular interactions—hydrogen and hydrophobic bonds shown as green and pink/purple dashed lines, respectively; (b) 2D plot of interactions between 5-methyl-3-furancarboxylic acid and key residues of PaDsbA1 generated by BIOVIA Discovery Studio visualizer.
Figure 4. (a) Docked pose of 5-methyl-3-furancarboxylic acid in the PaDsbA1 binding site showing molecular interactions—hydrogen and hydrophobic bonds shown as green and pink/purple dashed lines, respectively; (b) 2D plot of interactions between 5-methyl-3-furancarboxylic acid and key residues of PaDsbA1 generated by BIOVIA Discovery Studio visualizer.
Antibiotics 09 00911 g004
Antibiotics 09 00911 g005 550
Figure 5. (a) Docked pose of methylglyoxal in the PaDsbA1 binding site showing molecular interactions—hydrogen and hydrophobic bonds shown as green and pink/purple dashed lines, respectively; (b) 2D plot of interactions between methylglyoxal and key residues of PaDsbA1 generated by BIOVIA Discovery Studio visualizer.
Figure 5. (a) Docked pose of methylglyoxal in the PaDsbA1 binding site showing molecular interactions—hydrogen and hydrophobic bonds shown as green and pink/purple dashed lines, respectively; (b) 2D plot of interactions between methylglyoxal and key residues of PaDsbA1 generated by BIOVIA Discovery Studio visualizer.
Antibiotics 09 00911 g005
Antibiotics 09 00911 g006 550
Figure 6. (a) Docked pose of 5-hydroxymethyl-2-furaldehyde in the PaDsbA1 binding site showing molecular interactions—hydrogen and hydrophobic bonds shown as green and pink/purple dashed lines, respectively; (b) 2D plot of interactions between 5-hydroxymethyl-2-furaldehyde and key residues of PaDsbA1 generated by BIOVIA Discovery Studio visualizer.
Figure 6. (a) Docked pose of 5-hydroxymethyl-2-furaldehyde in the PaDsbA1 binding site showing molecular interactions—hydrogen and hydrophobic bonds shown as green and pink/purple dashed lines, respectively; (b) 2D plot of interactions between 5-hydroxymethyl-2-furaldehyde and key residues of PaDsbA1 generated by BIOVIA Discovery Studio visualizer.
Antibiotics 09 00911 g006
Table
Table 1. Molecular interactions of Manuka (M)/Heather (H) honey constituents showing the best ligand efficiencies for PaDsbA1 1.
Table 1. Molecular interactions of Manuka (M)/Heather (H) honey constituents showing the best ligand efficiencies for PaDsbA1 1.
Ligand (Origin)Docking Score (kcal/mol)Ligand EfficiencyInteracting ResiduesDistance (Å)CategoryType
Benzoic acid (M, H)−5.40.60Tyr1482.408H-BondConventional
Pro201.778H-Bond Conventional
Leu1443.456HydrophobicPi-Sigma
Val305.365HydrophobicPi-Alkyl
Val614.581HydrophobicPi-Alkyl
Leu634.592HydrophobicPi-Alkyl
5-methyl-3-furan-carboxylic acid (M)−5.10.57Tyr1482.414H-BondConventional
Pro201.953H-BondConventional
Val614.650HydrophobicAlkyl
Leu634.838HydrophobicAlkyl
Leu1444.654HydrophobicAlkyl
Val614.713HydrophobicAlkyl
Leu634.688HydrophobicAlkyl
Methyl-glyoxal (M)−2.80.56Ser222.996H-BondConventional
Ser223.512H-BondCarbon H-Bond
Ser223.509H-BondCarbon H-Bond
5-hydroxy-methyl-2-furaldehyde (M)−5.00.56Tyr1482.343H-BondConventional
Pro201.922H-BondConventional
Ser223.508H-BondCarbon H-Bond
Ser223.487H-BondCarbon H-Bond
Val304.982HydrophobicAlkyl
Val614.529HydrophobicAlkyl
Leu634.728HydrophobicAlkyl
Leu1444.541HydrophobicAlkyl
1 The control had a docking score of −6.1 kcal/mol and a ligand efficiency of 0.41.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Shirlaw, O.; Billah, Z.; Attar, B.; Hughes, L.; Qasaymeh, R.M.; Seidel, V.; Efthimiou, G. Antibiofilm Activity of Heather and Manuka Honeys and Antivirulence Potential of Some of Their Constituents on the DsbA1 Enzyme of Pseudomonas aeruginosa. Antibiotics 2020, 9, 911. https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics9120911

AMA Style

Shirlaw O, Billah Z, Attar B, Hughes L, Qasaymeh RM, Seidel V, Efthimiou G. Antibiofilm Activity of Heather and Manuka Honeys and Antivirulence Potential of Some of Their Constituents on the DsbA1 Enzyme of Pseudomonas aeruginosa. Antibiotics. 2020; 9(12):911. https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics9120911

Chicago/Turabian Style

Shirlaw, Oscar, Zara Billah, Baraa Attar, Lisa Hughes, Rana M. Qasaymeh, Veronique Seidel, and Georgios Efthimiou. 2020. "Antibiofilm Activity of Heather and Manuka Honeys and Antivirulence Potential of Some of Their Constituents on the DsbA1 Enzyme of Pseudomonas aeruginosa" Antibiotics 9, no. 12: 911. https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics9120911

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop