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. 2021 Jan 11;10:14. [Version 1] doi: 10.12688/f1000research.27915.1

Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa

Dina Auliya Amly 1, Puspita Hajardhini 1, Alma Linggar Jonarta 2, Heribertus Dedy Kusuma Yulianto 3, Heni Susilowati 2,a
PMCID: PMC8424461  PMID: 34540201

Abstract

Background: Pseudomonas aeruginosa, a multidrug resistant Gram-negative bacterium, produces pyocyanin, a virulence factor associated with antibiotic tolerance. High concentrations of royal jelly have an antibacterial effect, which may have the potential to overcome antibacterial resistance. However, in some cases, antibiotic tolerance can occur due to prolonged stress of low-dose antibacterial agents. This study aimed to investigate the effect of subinhibitory concentrations of royal jelly on bacterial growth and pyocyanin production of P. aeruginosa.

Methods:Pseudomonas aeruginosa ATCC ® 10145™ and clinical isolates were cultured  in  BHI media for 18 hours followed by optical density measurements at 600 nm wavelength to determine minimum inhibitory concentration (MIC). After 36 hours of incubation, pyocyanin production was observed by measuring the absorbance at 690 nm. Pyocyanin concentrations were calculated using extinction coefficient 4310 M -1cm -1.

Results: Results of the MIC tests of both strains were 25%. The highest production of pyocyanin was observed in the subinhibitory concentration group 6.25%, which gradually decreased along with the decrease of royal jelly concentration. Results of one-way ANOVA tests differed significantly in pyocyanin production of the two strains between the royal jelly groups. Tukey HSD test showed concentrations of 12.5%, 6.25%, and 3.125% significantly increased pyocyanin production of ATCC ® 10145™, and the concentrations of 12.5% and 6.25% significantly increased production of the clinical isolates.

Conclusions: This study concluded royal jelly concentrations of 25% or above could inhibit bacterial growth; however, only the concentrations of 12.5% and 6.25% could increase pyocyanin production in P. aeruginosa, both in ATCC ® 10145™ and clinical isolates. In conclusion, it is advisable to determine the appropriate concentration of royal jelly to obtain beneficial virulence inhibiting activity.

Keywords: royal jelly, antibacterial effect, Pseudomonas aeruginosa, pyocyanin

Introduction

Pseudomonas aeruginosa ( P. aeruginosa) is one of the Gram-negative bacilli bacteria which causes nosocomial infections that can be fatal, especially in immunocompromised patients 1– 3 . These bacteria are often found in the dental unit waterlines which allows the transmission of these bacteria into the oral cavity 4. As an opportunist pathogen, P. aeruginosa is also frequently involved in oral infections, such as necrotizing ulcerative gingivitis, periodontitis, and mandibular osteomyelitis 5– 7 . Although the mechanism is not clear yet, its presence in the oral cavity has been shown to result in systemic infections, such as nosocomial pneumonia 8.

Based on the reports from several clinical cases, the infection caused by P. aeruginosa bacteria can be fatal. Treatment of P. aeruginosa infection is sometimes ineffective, which is closely related to the number of virulence factors possessed by the bacteria 9. The bacterial cell surface components and some secretory products are important virulence factors of P. aeruginosa, one of which is pyocyanin 10. Pyocyanin is a cytotoxic pigment from the Phenazine group of compounds that can facilitate biofilm development, cause pro-inflammatory effects, and result in host cell death 11.

The resistance of P. aeruginosa to various spectrums of antibiotics creates difficulties in handling the infection it causes 12. It has been reported recently that the administration of antibiotics below the minimum inhibitory concentration (MIC) can cause specific bacterial responses, such as an increase in pyocyanin production in P. aeruginosa. PAO1 and P14 are the attempts by the bacteria to survive under antibiotic stress 13. This certainly motivates researchers to further analyze the infection they cause, and find the appropriate antibiotic concentration or dose to overcome the problem.

Royal jelly is a natural bee product that has the potential to be developed to overcome antibiotic resistance. Royal jelly has anti-inflammatory, antibacterial, and antioxidant effects 14. Royal jelly proteins, such as Jelleine, major royal jelly protein-1 (MRJP1), and royalicin are known to have antibacterial effects against P. aeruginosa. Major royal jelly protein-1 and Jelleine can interfere with the permeability of the outer membrane of the cell, causing the loss of vital contents of bacterial cells, which in turn causes cell death. Cationic antimicrobial peptides, such as royalicin, are known to also interfere with cell membrane permeability in various Gram-positive and Gram-negative bacteria, such as P. aeruginosa 15– 17 . Results of previous studies have shown that royal jelly can inhibit the growth of P. aeruginosa. In this study, royal jelly showed inhibition of the growth of P. aeruginosa ATCC ® 27853™ 18. In addition, it has also been known that royal jelly in various concentrations can inhibit the nonspecific attachment of P. aeruginosa ATCC ® 27853™ 19, but so far, the effect of the subinhibitory concentration of royal jelly against these bacteria is unknown. Furthermore, as pyocyanin is an indicator of the pathogenicity of P. aeruginosa strains, the aim of this study was to determine the effect of subinhibitory royal jelly concentration on pyocyanin production in representative strains of a high level pyocyanin-producer P. aeruginosa ATCC ® 10145™ and clinical isolates.

Methods

This in vitro laboratory experimental research was done at the Integrated Research Laboratory of the Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta. All research procedures have been approved by the Ethics Committee of the Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta (No. 00393/KKEP/ FKG-UGM/EC/2020).

The royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia. This product is produced from Apis mellifera bees that have been identified previously 19. Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4°C). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant was filtered using 0.45 µm millipore to produce 55% royal jelly. Furthermore, royal jelly was stored at a temperature of 4–8°C 20.

Pseudomonas aeruginosa ATCC ® 10145™ (Thermo Scientific) was obtained from the Integrated Research Laboratory of the Faculty of Dentistry, Universitas Gadjah Mada. A clinical isolate of P. aeruginosa derived from patient sputum was obtained from the Laboratory of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada. Both of these strains were each inoculated in Luria Bertani broth and incubated at 37°C for 24 hours. After that, the culture was centrifuged at 3000 rpm for 15 minutes and then resuspended using 0.98% NaCl to obtain a bacterial concentration equivalent to 1.5 × 10 5 CFU/ml.

Measurement of the effect of royal jelly on the viability of P. aeruginosa

A sterile 55% w/v royal jelly solution was diluted in brain heart infusion (BHI; Himedia Laboratories) broth to obtain a concentration of 50% and then serial dilution was performed in 96 well microplates. A total of 5 µl of the P. aeruginosa ATCC ® 10145™ suspension or clinical isolate bacteria (1.5 × 10 5 CFU/ml) was inoculated in all groups, except the groups that had been determined as blanks (blanko). The culture was then incubated at 37°C for 18 hours. After that, the microplate was scanned using the Spark® Multimode Microplate Reader (Tecan trading AG) to measure optical density (OD) using a 600 nm wavelength. The percentage of bacterial viability inhibition was determined based on the OD value of the treatment group against the control.

Analysis of the effect of royal jelly on pyocyanin production

Royal jelly solution was diluted into sterile BHI broth to get the concentration of 12.5%, 6.25%, 3.125%, 1.56%, 0.78%, 0.39%, 0.19%, and 0.098% w/v. Both strains of P. aeruginosa were cultured on BHI broth containing various concentrations of royal jelly as treatment and BHI broth only as a blank (blanko). The cultures were incubated at 37°C for 36 hours, then the pyocyanin production of each strain was observed visually, which appeared green in the culture supernatant. The pyocyanin concentration was further quantified using previously published methods 21. Briefly, after 36 hours of incubation, the culture supernatant was transferred to a sterile tube and centrifuged at a rate of 10,000 g for 30 minutes. The supernatant was filtered using a 0.45 µm Millipore filter and transferred to 96 new well microplates. The absorbance value of the supernatant containing pyocyanin was measured at a wavelength of 690 nm, then the pyocyanin concentration was calculated using the following equation 21.

Concentration of pyocyanin = A 690 nm (A 690 nm of sample - A 690 nm of blank) / ε * d

ε = extinction coefficient (pyocyanin at A 690 nm = 4310 M -1cm -1)

d = path length (0.23 cm for 96 well microplate)

Statistical analysis

The data in this study were presented as the percentage of bacterial viability and pyocyanin concentration in the P. aeruginosa culture supernatant. All data were tested for normality using the Shapiro-Wilk and the Levene Test for homogeneity using SPSS Statistic v20. Furthermore, one-way ANOVA and Games-Howell parametric analysis were performed for bacterial cell viability data; and parametric one-way ANOVA followed by Tukey HSD on pyocyanin concentration data.

Results

Antibacterial activity of royal jelly against P. aeruginosa

The antibacterial activity of royal jelly against the two strains of P. aeruginosa is shown in Figure 1. Data on the percentage of bacterial growth inhibition shows normal distribution data ( p>0.05), but has a non-homogeneous variant ( p<0.05). One-way ANOVA showed a significant difference in the percentage of growth inhibition in P. aeruginosa ATCC ® 10145™ ( p = 0.000) and P. aeruginosa clinical isolate ( p = 0.000) between royal jelly treatment groups and negative control. In this study, it was proven that royal jelly can inhibit the viability of both P. aeruginosa strains starting from a concentration of 25%. The results of the multi-comparison analysis showed that there was no significant difference between the concentrations of 25% and 50% and significant differences were identified between the concentrations of 25% and 50% with 12.5% to 0.098% in both strains. It can be concluded that the MIC for both strains is 25%.

Figure 1. Percentage of inhibition of growth of P. aeruginosa bacteria (1.5 × 10 5 CFU/well).

Figure 1.

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Bacterial cultures were incubated with varying concentrations of royal jelly for 18 hours at 37°C. Royal jelly 50% and 25% inhibit bacterial growth. The difference is based on the results of the Games-Howell analysis at the significance value (*) p <0.05. ATCC ® 10145™ ( a) strain; clinical isolate ( b).

Exposure to subinhibitory royal jelly concentrations induced increased pyocyanin production in P. aeruginosa

Pyocyanin was identified as green in culture supernatant P. aeruginosa ATCC ®10145™ and clinical isolate. After 36 hours of incubation, pyocyanin production was increased in the stimulated culture group with subinhibitory concentrations below 25%. The intensity of green color in the culture medium increased with the increase in the concentration of royal jelly ( Figure 2). The change in the color intensity of the culture supernatant was consistent with the results of the pyocyanin concentration measurement.

Figure 2. Pyocyanin was identified from the green color of the P. aeruginosa culture supernatant after 36 h incubation at 37°C.

Figure 2.

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P. aeruginosa bacteria (1.5 × 10 5 CFU/well) ATCC ® 10145™ ( a); clinical isolate ( b). Royal jelly 12.5% (A); 6.25% (B); 3,125% (C); 1.56% (D); 0.78% (E); 0.39% (F); 0.19% (G); 0.098% (H); 0% (I); No treatment (J).

Pyocyanin concentration data in each royal jelly treatment group and negative control were the results of experiments on triplicate cultures. Figure 3 shows the average pyocyanin concentration for each group. The highest average pyocyanin concentration was identified in P. aeruginosa ATCC ® 10145™ induced by royal jelly with a concentration of 6.25%, which was 23.59 μM, while the lowest mean was identified in clinical isolates of P. aeruginosa without exposure to royal jelly, which was 0.7 μM. The pyocyanin concentration of P. aeruginosa ATCC ® 10145™ was seen to be higher than clinical isolate in the same concentration in all treatment groups.

Figure 3. Pyocyanin production in P. aeruginosa bacteria (1.5 × 10 5 CFU/well) after 36 hours of exposure to various concentrations of royal jelly.

Figure 3.

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Pyocyanin production increased due to exposure to sub-inhibitory royal jelly concentrations of 6.25% and decreased at lower royal jelly concentrations. The difference was based on the results of the Tukey-HSD analysis at the significance value (*) p <0.05. ATCC ® 10145™ ( a) strain; ( b) clinical isolate.

Pyocyanin concentration data both on ATCC ® 10145™ and clinical isolate in all groups were normally distributed ( p>0.05) and homogeneous ( p>0.05). There was a significant difference in the concentration of pyocyanin ATCC ® 10145™ ( p = 0.000) and clinical isolate ( p = 0.000) between the treatment groups. The results of multiple comparison analysis of Tukey-HSD on P. aeruginosa cultures of ATCC ® 10145™ showed a significant difference between the royal jelly groups with concentrations of 0% with 12.5%, 6.25%, and 3.125%. In addition, a significant difference in pyocyanin concentrations in clinical isolate was found between the 0% royal jelly group with 12.5% and 6.25%.

Discussion

The antibacterial effect of royal jelly has been widely reported by previous researchers 15– 17 . The ability of royal jelly to inhibit the growth of P. aeruginosa is thought to be related to the variety and concentration of its antibacterial protein. Royal jelly components that have been identified as having antibacterial activity are major royal jelly protein-1 (MRJP-1), Jelleine I–III, royalicin, and 10-hydroxy-2-decenoic (10-HDA) 15, 17, 22.

This study showed that royal jelly concentrations of 25% and 50% had antibacterial activity against P. aeruginosa ATCC ® 10145™ and clinical isolate. The results of this observation are different from previous studies that showed P. aeruginosa growth could be inhibited at concentrations >50% 18. This difference is thought to be closely related to differences in geographical location, botanical origin, climate, and storage conditions of royal jelly, which affect the antibacterial component of royal jelly 23. Previous studies have shown that royal jelly originating from different geographic and botanical locations affects the quantity of 10-HDA. Royal jelly originating from tropical climates is reported to contain lower concentrations of 10-HDA than cold climates 23. The higher temperature and longer storage time also resulted in a significant reduction in the quantity of MRJP1 24. However, the bacterial strains studied probably also had an effect, as previously reported there was a variable response between clinical isolates and standard bacteria 18, 19.

Pyocyanin is an indicator of the pathogenicity of P. aeruginosa. To our knowledge, this study report is the first to demonstrate a dualism effect of royal jelly on P. aeruginosa. The subinhibitory concentration of royal jelly amplify the effect of an autoinducer. It was able to increase the production of pyocyanin in ATCC ® 10145™ and clinical isolates to protect and maintain their survival 13. The pyocyanin concentration in the ATCC ® 10145™ strain appeared to be significantly higher than the clinical isolates. This observation is in accordance with previous studies that found the ATCC ® 10145™ strain produced more pyocyanin than the clinical isolate strains from active ulcerative keratitis patients 25. The presence of phzM and phzS genes was thought to affect the concentration of pyocyanin produced 26. This was proven by previous studies that the phzM and phzS gene expression of multidrug resistance (MDR) clinical isolate P. aeruginosa was lower leading to less pyocyanin production than non-MDR isolates and PAO1 strains 27. Some clinical isolates were also reported not to have the genes so that these bacteria cannot produce pyocyanin 26. Other research results also showed that the pyocyanin concentration of ATCC ® 10145™ strains is higher than that of PAO1 and PA14 strains after incubation for 60 hours 28. It is estimated that ATCC ® 10145™ is one of the strong pyocyanin producing strains. However, other virulence factors possessed by this strain were lower than the clinical isolate strains so that they were considered less virulent 25.

Various virulence factors, including pyocyanin are generally associated with the quorum sensing mechanism 29. Quorum sensing refers to the communication process between microbial cells using autoinducer molecules 30. One of the autoinducer molecules that plays an important role in the regulation of pyocyanin production is the pseudomonas quinolone signal (PQS). Mutation of the PQS gene results in reduced pyocyanin production 31. When bacterial cells are exposed to exogenic stress, such as an antibacterial agent that can threaten their survival, the bacteria immediately respond to the stimulus by inducing the production of PQS which is responsible for activating various genes involved in the production of virulence factors, including pyocyanin 29, 32. Although the effect of royal jelly subinhibitor concentration on this autoinducer molecule is not yet known, several studies have reported that the increase in pyocyanin production is closely related to the effect of subinhibitor antibiotics that increase PQS gene expression 33. It is thought that this is the cause of increased pyocyanin production at subinhibitory concentrations.

The increase in pyocyanin production in P. aeruginosa bacteria will have implications for the mechanism of bacterial attachment and biofilm formation. Apart from its production, which is closely related to the quorum sensing mechanism, pyocyanin is also a signaling factor in the quorum sensing process itself. This was identified from the results of research on P. aeruginosa PAO1 and PA14 34. In addition, the increase in pyocyanin is likely to have an impact on the activity of bacteria to produce extracellular DNA (eDNA). Extracellular DNA is an important part of extracellular polymeric substance (EPS) which is the main component of the biofilm matrix. The increase in EPS production is very beneficial for the bacterial attachment process and subsequently the formation of biofilms. Pyocyanin can induce eDNA production in low level pyocyanin-producer strains, PAO1 and pyocyanin-deficient strains, PA14. In this study, it was proven that pyocyanin caused an increase in the production and release of eDNA, which is the main component in forming and stabilizing bacterial biofilms 35.

The increase in pyocyanin production induced by subinhibitory royal jelly concentrations in P. aeruginosa ATCC ® 10145™ and clinical isolates in this study is an interesting phenomenon. Although, subinhibitory royal jelly concentrations were not effective in inhibiting the growth of these bacteria, on the other hand, they increased production of pyocyanin virulence factors. This has inspired the alleged biphasic nature of royal jelly which has antibacterial potential, but at different exposure concentrations, it can induce the production of P. aeruginosa bacteria virulence factors. This phenomenon leads us to think that researchers, as well as medical practitioners, should be careful in determining the concentration of royal jelly for its antibacterial research purposes or its therapeutic potential. This of course requires further research on the mechanisms associated with bacterial response to subinhibitory concentrations of royal jelly.

Conclusions

Royal jelly at a concentration of 25% was only able to inhibit the growth of P. aeruginosa bacteria, but at subinhibitory concentrations it could increase pyocyanin production in P. aeruginosa strain ATCC ® 10145™ and clinical isolate. Based on the results of this study, we suggest selecting the appropriate dose or concentration for the purpose of inhibiting the growth and production of P. aeruginosa virulence factors.

Data availability

Underlying data

Figshare: Pseudomonas aeruginosa pyocyanin, https://doi.org/10.6084/m9.figshare.13247429.v1 36.

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgements

Researchers appreciate the help of Bunga Artika as a laboratory assistant who helped in this research.

Funding Statement

Part of this research was supported by funding from the Ministry of Research, Technology and Higher Education, Republic of Indonesia, The Excellence Basic Research [Grant No. 26023/ UN1.DITLIT/DIT.LIT/LT/2019] to Heni Susilowati.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

[version 1; peer review: 1 approved with reservations

References

  • 1.Corrin B, Nicholson AG: Pathology of the Lungs. 3 rd ed. London: Churchill Livingstone. Elsevier. 2011;177. 10.1016/C2009-0-43318-X [DOI] [Google Scholar]
  • 2.Migiyama Y, Yanagihara K, Kaku N, et al. : Pseudomonas aeruginosa bacteremia among immunocompetent and immunocompromised patients: relation to initial antibiotic therapy and survival. Jpn J Infect Dis. 2016;69(2):91–96. 10.7883/yoken.JJID.2014.573 [DOI] [PubMed] [Google Scholar]
  • 3.Wolska K, Kot B, Jakubczak A: Phenotypic and genotypic diversity of Pseudomonas aeruginosa strains isolated from hospitals in Siedlce (Poland). Braz J Microbil. 2012;43(1):274–282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ouellet MM, Leduc A, Nadeau C, et al. : Pseudomonas aeruginosa isolates from dental unit waterlines can be divided in two distinct groups, including one displaying phenotypes similar to isolates from cystic fibrosis patients. Front Microbiol. 2015;5:802. 10.3389/fmicb.2014.00802 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Jandial A, Mishra K, Panda A, et al. : Necrotising ulcerative gingivitis: a rare manifestation of Pseudomonas infection. Indian J Hematol Blood Transfus. 2018;34(3):578–580. 10.1007/s12288-018-0927-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Colombo APV, Magalhaes C, Hartenbach FAR, et al. : Periodontal disease associated biofilm : a reservoir for pathogens of medical importance. Microb Pathog. 2016;94:27–34. 10.1016/j.micpath.2015.09.009 [DOI] [PubMed] [Google Scholar]
  • 7.Pappalardo S, Tanteri L, Brutto D, et al. : Mandibular osteomyelitis due to Pseudomonas aeruginosa. Case report. Minerva Stomatol. 2008;57(6):323–329. [PubMed] [Google Scholar]
  • 8.Caldas R, Boisrame S: Upper aero-digestive contamination by Pseudomonas aeruginosa and implications in Cystic Fibrosis. J Cyst Fibros. 2015;14(1):6–15. 10.1016/j.jcf.2014.04.008 [DOI] [PubMed] [Google Scholar]
  • 9.Macin S, Akarca M, Sener B, et al. : Comparison of virulence factors and antibiotic resistance of Pseudomonas aeruginosa strains isolated from patients with and without cystic fibrosis. Rev Rom Med Lab. 2017;25(4):327–334. 10.1515/rrlm-2017-0027 [DOI] [Google Scholar]
  • 10.Khalifa ABH, Moissenet D, Thien HV, et al. : Virulence factor in Pseudomonas aeruginosa: mechanisms and modes of regulation. Ann Biol Clin (paris). 2011;69(4):393–403. 10.1684/abc.2011.0589 [DOI] [PubMed] [Google Scholar]
  • 11.Hall S, McDermott C, Anoopkumar-Dukie S, et al. : Cellular effects of pyocyanin, a secreted virulence factor of Pseudomonas aeruginosa. Toxins (Basel). 2016;8(8):236. 10.3390/toxins8080236 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Nguyen L, Garcia J, Gruenberg K, et al. : Multidrug-resistant Pseudomonas infections: hard to treat, but hope on the horizon? Curr Infect Dis Rep. 2018;20(8):23. 10.1007/s11908-018-0629-6 [DOI] [PubMed] [Google Scholar]
  • 13.Zhu K, Chen S, Sysoeva TA, et al. : Universal antibiotic tolerance arising from antibiotic-triggered accumulation of pyocyanin in Pseudomonas aeruginosa. PLoS Biol. 2019;17(12): e3000573. 10.1371/journal.pbio.3000573 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Fratini F, Cilia G, Mancini S, et al. : Royal jelly: an ancient remedy with remarkable antibacterial properties. Microbiol Res. 2016;192:130–141. 10.1016/j.micres.2016.06.007 [DOI] [PubMed] [Google Scholar]
  • 15.Brudzynski K, Sjaarda C, Lannigan R: MRJP1- containing glycoproteins isolated from honey, a novel antibacterial drug candidate with broad spectrum activity against multi-drug resistant clinical isolates. Front Microbiol. 2015;6:711. 10.3389/fmicb.2015.00711 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Fontana R, Mendes MA, De Souza BM, et al. : Jelleines: a family of antimicrobial peptides from the royal jelly of honeybees ( Apis mellifera). Peptides. 2004;25(6):919–928. 10.1016/j.peptides.2004.03.016 [DOI] [PubMed] [Google Scholar]
  • 17.Bilikova K, Huang SC, Lin IP, et al. : Structure and antimicrobial activity relationship of royalisin, an antimicrobial peptide from royal jelly of Apis mellifera. Peptides. 2015;68:190–196. 10.1016/j.peptides.2015.03.001 [DOI] [PubMed] [Google Scholar]
  • 18.Boukraa L: Additive activity of royal jelly and honey against Pseudomonas aeruginosa. Altern Med Rev. 2008;13(4):330–333. [PubMed] [Google Scholar]
  • 19.Hartono SK, Haniastuti T, Susilowati H, et al. : The effect of in vitro royal jelly provision on adhesion of Pseudomonas aeruginosa. Maj Kedokt Gigi Indones. 2019;1(1):1-5. 10.22146/majkedgiind.30221 [DOI] [Google Scholar]
  • 20.Susilowati H, Murakami K, Yumoto H, et al. : Royal jelly inhibits Pseudomonas aeruginosa adherence and reduces excessive inflammatory responses in human epithelial cells. Biomed Res Int. 2017;2017:3191752. 10.1155/2017/3191752 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Price-Whelan A, Dietrich LEP, Newman DK: Pyocyanin alters redox homeostasis and carbon flux through central metabolic pathways in Pseudomonas aeruginosa PA14. J Bacteriol. 2007;189(17):6372–6381. 10.1128/JB.00505-07 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Han B, Fang Y, Feng M: In-depth phosphoproteomic analysis of royal jelly derived from western and eastern honeybee species. J Proteome Res. 2014;13(12):5928–5943. 10.1021/pr500843j [DOI] [PubMed] [Google Scholar]
  • 23.Wei WT, Hu YQ, Zheng HQ, et al. : Geographical influences on content of 10-hydroxy-trans-2-decenoic acid in royal jelly in China. J Econ Entomol. 2013;106(5):1958–1963. 10.1603/ec13035 [DOI] [PubMed] [Google Scholar]
  • 24.Li JK, Feng M, Zhang L, et al. : Proteomics analysis of major royal jelly protein changes under different storage conditions. J Proteome Res. 2008;7(8):3339–3353. 10.1021/pr8002276 [DOI] [PubMed] [Google Scholar]
  • 25.Sewell A, Dunmire J, Wehmann M, et al. : Proteomic analysis of keratitis-associated Pseudomonas aeruginosa. Mol Vis. 2014;20:1182–1191. [PMC free article] [PubMed] [Google Scholar]
  • 26.Samad A, Ahmed T, Rahim A, et al. : Antimicrobial susceptibility patterns of clinical isolates of Pseudomonas aeruginosa isolated from patients of respiratory tract infections in a Tertiary Care Hospital, Peshawar. Pak J Med Sci. 2017;33(3):670–674. 10.12669/pjms.333.12416 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Fuse K, Fujimura S, Kikuchi T, et al. : Reduction of virulence factor pyocyanin production in multidrug-resistant Pseudomonas aeruginosa. J Infect Chemother. 2013;19(1): 82–88. 10.1007/s10156-012-0457-9 [DOI] [PubMed] [Google Scholar]
  • 28.Rada B, Lekstrom K, Damian S, et al. : The Pseudomonas toxin pyocyanin inhibits the dual oxidase- based antimicrobial system as it imposes oxidative stress on airway epithelial cells. J Immunol. 2008;181(7):4883–4893. 10.4049/jimmunol.181.7.4883 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Lin J, Cheng J, Wang Y, et al. : The Pseudomonas Quinolone signal (PQS): not just for quorum sensing anymore. Front Cell Infect Microbiol. 2018;8:230. 10.3389/fcimb.2018.00230 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Zhang W, Li C: Exploiting quorum sensing interfering strategies in gram-negative bacteria for the enhancement of environmental applications. Front Microbiol. 2016;6:1535. 10.3389/fmicb.2015.01535 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Bala A, Kumar L, Chhibber S, et al. : Augmentation of virulence related traits of pqs mutants by Pseudomonas quinolone signal through membrane vesicles. J Basic Microbiol. 2014;55(5):566–578. 10.1002/jobm.201400377 [DOI] [PubMed] [Google Scholar]
  • 32.Bru J, Rawson B, Trinh C, et al. : PQS produced by the Pseudomonas aeruginosa stress response repels swarms away from bacteriophage and antibiotics. J Bacteriol. 2019;201(23):e00383–19. 10.1128/JB.00383-19 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Cummins J, Reen FJ, Baysse C, et al. : Subinhibitor concentrations of the cationic antimicrobial peptide colistin induce the pseudomonas quinolone signal in Pseudomonas aeruginosa. Microbiology(Reading). 2009;155(Pt 9):2826–2837. 10.1099/mic.0.025643-0 [DOI] [PubMed] [Google Scholar]
  • 34.Dietrich LE, Price-Whelan A, Petersen A, et al. : The penazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa. Mol Microbiol. 2006;61(5):1308–1321. 10.1111/j.1365-2958.2006.05306.x [DOI] [PubMed] [Google Scholar]
  • 35.Das T, Manefield M: Pyocyanin promotes extracellular DNA release in Pseudomonas aeruginosa. PLoS One. 2012;7(10):e46718. 10.1371/journal.pone.0046718 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Susilowati H, Amly DA, Yulianto HDK, et al. : Pseudomonas aeruginosa pyocyanin. figshare.Figure.2020. 10.6084/m9.figshare.13247429.v1 [DOI]
F1000Res. 2021 Feb 4. doi: 10.5256/f1000research.30869.r77077

Reviewer response for version 1

Theerthankar Das 1

The intention of this study is good and to enlighten the antibacterial and anti-virulence effect of royal jelly in P. aeruginosa.

To be more conclusive this study needs more work:

  1. Its better to use chloroform-HCl (standard and more popular) assay to quantify pyocyanin yield - its more rigorous than just taking absorbance at 690nm of pyocyanin (broad peak).

  2. Biofilm (Crystal violet for biomass quantification) study needs to be done to ensure impact of royal jelly/pyocyanin on biofilm development.

  3. Confocal microscopy will be of great help to show biofilm architecture.

Specific comments:

  • Why solubilized royal jelly is centrifuged and supernatant is only taken? What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?

  • At 25% royal jelly we see only 60% growth inhibition for both bacterial strains. Its not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Biofilms, antimicrobial agents

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

F1000Res. 2021 Jun 7.
Heni Susilowati 1

We thank you for the thorough and careful review of our manuscript. We apologize for our delay in revisions due to the various obstacles we face regarding the availability of laboratory facilities and access during the Covid-19 pandemic.

The following are our responses to the suggestions and specific comments.

The response  to comment 1 (quantification of pyocyanin)

We measured the concentration of pyocyanin contained in the culture supernatant of P. aeruginosa. This condition is in accordance with the previous research conducted by Price-Whelan et al. (2007). Indeed, we only measured the absorbance value of the supernatant filtrate of the bacterial culture at a wavelength of 690 nm, but the pyocyanin concentration was then known by calculations using a formula published by previous researchers (Price-Whelan et al, 2007). The constant value of 4310 M -1cm -1 for the absorbance at 690 nm has been determined based on the research of Dietrich et al. 2006. Since our experimental conditions match the experimental conditions in both studies, both of which are published in outstanding journals, we feel it is sufficient to refer to their methods.

The response  to comment 2 ( Biofilm [Crystal violet for biomass quantification] study)

We have conducted additional experiments to detect the association between pyocyanin production in bacteria exposed to royal jelly and biofilm formation using the static microtiter plate biofilm assay (crystal violet staining) method. To analyze the relationship between pyocyanin production and biofilm mass production, we have used P. aeruginosa ATCC 10145 as this strain is more responsive in producing pyocyanin when compared to the clinical isolate strains we previously use. The results showed that the detectable biofilm mass significantly increased in bacterial cultures exposed to 12.5% ​​royal jelly extract. Meanwhile, cultures treated with 25% royal jelly did not show any biofilm formation. The 6.25% royal jelly extract and the lower concentrations induced biofilm mass formation but at a lower quantity than 12.5% ​​royal jelly. This evidence suggests a relationship between royal jelly concentration, pyocyanin production, and biofilm mass formation. The subinhibitory concentration (12.5%, 6.23%) of the royal jelly extract induced more pyocyanin production and biofilm mass formation rather than the higher concentrations.

Related changes were in the Methods, Results, and Conclusion of the Abstract, paragraph 6 of the Methods, paragraph 5 of Results, Figure 4, and line 5 of paragraph 6 on the Discussion section in the manuscript.

The response  to comment 3 (microscopy of biofilm architecture)

In order to complement the data regarding the effect of royal jelly on the mass formation of biofilms, we have carried out observations of the microscopic architecture of P. aeruginosa ATCC 10145 biofilms using scanning electron microscopy, since we could not access the confocal microscopy method at our university. We chose secondary electron (SE) mode rather than a backscattered electron (BSE) to investigate biofilm in a three-dimensional perspective. The results of observations on representative samples showed that in the culture exposed to 25% royal jelly extract there was no biofilm mass deposition, while the group exposed to 12.5% royal jelly showed a larger density of biofilm than the 6.25% royal jelly group. Chlorhexidine 0.2% control showed total inhibition of biofilm mass formation.

Methods, results, discussion, and related references have been added to the manuscript on Methods, Results, and Conclusion in the Abstract, paragraph 7 of the Methods section, paragraph 6-7 of the Results section, and line 5 in paragraph 6 of the Discussion section.

Specific comments:

1. Why solubilized royal jelly is centrifuged and supernatant is only taken?

Answer: The extraction method using phosphate buffer saline (Hu et al, 2019) was chosen in order to dissolve major royal jelly protein 1 (MRJP1), the active components that are antibacterial or anti-adhesion. It is known that MRJP1 is a hydrosoluble protein dissolved in PBS, which is higher in supernatants than in pellets (Gismondi et al., 2017).

What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?

After centrifugation, there were indeed pellets, and it has been found that the pellets contain more liposoluble proteins than supernatants. To ensure that the compounds contained in the PBS royal jelly extract supernatant, further research is needed; However, from the research of Furusawa et al. (2016), it was found that MRJP1 was contained in the supernatant of PBS royal jelly extract in quite a large amount, reaching 27.6%.

The active components that are antibacterial or anti-adhesion in the supernatant of royal jelly extract in this study cannot be explained with certainty, but the royal jelly used in this study was thought to mainly contain MRJP1.

We add this explanation to paragraph 2 of the Discussion section. Additional sources of literature have been written in the reference list numbers 37, 38, and 39.

2. At 25% royal jelly, we see only 60% growth inhibition for both bacterial strains. It is not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.

Answer: We agree with the reviewer's comments regarding the MIC score. In clinical isolates, 25% royal jelly extract caused 60% death in the bacterial population; meanwhile, in the standard strain ( P. aeruginosa ATCC 10145), this concentration caused growth inhibition of 85%.

We have revised the first sentence in Methods of Abstract and two last sentences in the Results section.

F1000Res. 2021 Jun 11.
Heni Susilowati 1

We thank you for the thorough and careful review of our manuscript. 

The following are our responses to the suggestions and specific comments.

The response  to comment 1 (quantification of pyocyanin)

We measured the concentration of pyocyanin contained in the culture supernatant of P. aeruginosa. This condition is in accordance with the previous research conducted by Price-Whelan et al. (2007). Indeed, we only measured the absorbance value of the supernatant filtrate of the bacterial culture at a wavelength of 690 nm, but the pyocyanin concentration was then known by calculations using a formula published by previous researchers (Price-Whelan et al, 2007). The constant value of 4310 M -1cm -1 for the absorbance at 690 nm has been determined based on the research of Dietrich et al. 2006. Since our experimental conditions match the experimental conditions in both studies, both of which are published in outstanding journals, we feel it is sufficient to refer to their methods.

The response to comment 2 (Biofilm [Crystal violet for biomass quantification] study)

We have conducted additional experiments to detect the association between pyocyanin production in bacteria exposed to royal jelly and biofilm formation using the static microtiter plate biofilm assay (crystal violet staining) method. To analyze the relationship between pyocyanin production and biofilm mass production, we have used P. aeruginosa ATCC 10145 as this strain is more responsive in producing pyocyanin when compared to the clinical isolate strains we previously use. The results showed that the detectable biofilm mass significantly increased in bacterial cultures exposed to 12.5% royal jelly extract. Meanwhile, cultures treated with 25% royal jelly did not show any biofilm formation. The 6.25% royal jelly extract and the lower concentrations induced biofilm mass formation but at a lower quantity than 12.5% royal jelly. This evidence suggests a relationship between royal jelly concentration, pyocyanin production, and biofilm mass formation. The subinhibitory concentration (12.5%, 6.23%) of the royal jelly extract induced more pyocyanin production and biofilm mass formation rather than the higher concentrations.

Related changes were in the Methods, Results, and Conclusion of the Abstract, paragraph 6 of the Methods, paragraph 5 of Results, Figure 4, and line 5 of paragraph 6 on the Discussion section in the manuscript.

The response  to comment 3 (microscopy of biofilm architecture)

In order to complement the data regarding the effect of royal jelly on the mass formation of biofilms, we have carried out observations of the microscopic architecture of P. aeruginosa ATCC 10145 biofilms using scanning electron microscopy, since we could not access the confocal microscopy method at our university. We chose secondary electron (SE) mode rather than a backscattered electron (BSE) to investigate biofilm in a three-dimensional perspective. The results of observations on representative samples showed that in the culture exposed to 25% royal jelly extract there was no biofilm mass deposition, while the group exposed to 12.5% royal jelly showed a larger density of biofilm than the 6.25% royal jelly group. Chlorhexidine 0.2% control showed total inhibition of biofilm mass formation.

Methods, results, discussion, and related references have been added to the manuscript on Methods, Results, and Conclusion in the Abstract, paragraph 7 of the Methods section, paragraph 6-7 of the Results section, and line 5 in paragraph 6 of the Discussion section.

Specific comments

a. Why solubilized royal jelly is centrifuged and supernatant is only taken?

Answer: The extraction method using phosphate buffer saline (Hu et al, 2019) was chosen in order to dissolve major royal jelly protein 1 (MRJP1), the active components that are antibacterial or anti-adhesion. It is known that MRJP1 is a hydrosoluble protein dissolved in PBS, which is higher in supernatants than in pellets (Gismondi et al., 2017).

What happens with pellets (are there any pellets found after centrifugation?). If yes - then how can we know that supernatant has all royal jelly?

Answer: After centrifugation, there were indeed pellets, and it has been found that the pellets contain more liposoluble proteins than supernatants. To ensure that the compounds contained in the PBS royal jelly extract supernatant, further research is needed; However, from the research of Furusawa et al. (2016), it was found that MRJP1 was contained in the supernatant of PBS royal jelly extract in quite a large amount, reaching 27.6%.

The active components that are antibacterial or anti-adhesion in the supernatant of royal jelly extract in this study cannot be explained with certainty, but the royal jelly used in this study was thought to mainly contain MRJP1.

We add this explanation to paragraph 2 of the Discussion section. Additional sources of literature have been written in the reference list numbers 37, 38, and 39.

b. At 25% royal jelly, we see only 60% growth inhibition for both bacterial strains. It is not MIC - you can call it LC50 (lethal dose to reduce at least 50% growth). MIC is when there is zero or 1-2% growth only.

Answer: We agree with the reviewer's comments regarding the MIC score. In clinical isolates, 25% royal jelly extract caused 60% death in the bacterial population; meanwhile, in the standard strain ( P. aeruginosa ATCC 10145), this concentration caused growth inhibition of 85%.

We have revised the first sentence in Methods of Abstract and two last sentences in the Results section.

F1000Res. 2021 Feb 1. doi: 10.5256/f1000research.30869.r77076

Reviewer response for version 1

Ariadna A Djais 1

This study examined royal jelly and  Apis mellifera bees as a base ingredient. These ingredients are antibacterial with pycocyanin as a pigment by the bacterium  Psedomonas aeroginosa. The  P. aeroginosa was selected because these bacteria are known as multidrug-resistant bacterium. This research is interesting and specific. In this study, the data was analyzed as an indicator of the difference in the contrast of the green pigment color, which was carried out by measuring at a wavelength of 690 nm.

In my opinion, this research needs to add more information regarding the methods chapters:

  1. It is necessary to add information about the stage of royal jelly making as the test material.

  2. It is necessary to add information about the bacterium identification technique which used by the researcher when confirming clinical isolates as  P. aeroginosa.

  3. Number of  replication of independent experiment conducted by the researcher.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

NA

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

F1000Res. 2021 Feb 14.
Heni Susilowati 1

Thank you for providing a review and suggestions for improving our manuscript. Below are our revisions to the manuscript according to the review comments.

1. Response to comments 1:

We explain the stages of preparing royal jelly on the manuscript, written in the Methods section (page 3) as follows:

Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May-October 2019. This product was obtained from the beekeeper Apis mellifera who lives in the randu trees ( Ceiba pentandra) and sono wood ( Dalbergia latifolia). Apis mellifera bee species have been identified through previous research19. Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4 oC). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant is taken and checked the pH. The results of pH measurements with a pH meter showed a pH of 3.79. Furthermore, the supernatant was filtered using 0.45 µm Millipore to produce 55% royal jelly.

2. Response to comment 2:

We write an explanation of how to identify bacteria in the Methods section (page 3), which is as follows:

Identification of the clinical isolate was carried out using API 2ONE (bioMérieux, Inc).

3. Response to comment 3:

Each experiment was carried out with 6 replications.

We write the revision on page 3 of the Methods section, on bacterial viability tests and pyocyanin detection.

F1000Res. 2021 Jun 11.
Heni Susilowati 1

Thank you for providing a review and suggestions for improving our manuscript.

Below are our revisions to the manuscript according to the review comments.

1. The response to comment 1

We explain the stages of preparing royal jelly on the manuscript, written in line 2-6 of paragraph 2 of the Methods section as follows:

Royal jelly used in this study was obtained from Nusukan, Surakarta, Central Java, Indonesia and harvested in May-October 2019. This product was obtained from the beekeeper Apis mellifera who lives in the randu trees ( Ceiba pentandra) and sono wood ( Dalbergia latifolia). Apis mellifera bee species have been identified through previous research (Hartono et al., 2019). Royal jelly 5.5 grams was dissolved in 10 ml of cold phosphate buffered saline (PBS), then homogenized using a magnetic stirrer (24 hours, 4 oC). The royal jelly solution was centrifuged (12,000 g, 45 minutes, 4°C), then the supernatant was taken and checked for the pH. The results of pH measurements using a pH meter showed a pH of 3.79. Furthermore, the supernatant was filtered using 0.45 µm Millipore to produce 55% royal jelly.

2. The response to comment 2

We have written an explanation of how to identify bacteria in line 5 of paragraph 3 the Methods section, which is as follows:

Identification of the clinical isolate was carried out using API 2ONE (BioMérieux, Inc).

3. Response   to comment 3

The bacterial viability assay experiment was carried out in quadruplicate, meanwhile, the pyocyanin experiments were performed in triplicate.

We write the revision in the last line of related paragraphs in the Methods section.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Citations

    1. Susilowati H, Amly DA, Yulianto HDK, et al. : Pseudomonas aeruginosa pyocyanin. figshare.Figure.2020. 10.6084/m9.figshare.13247429.v1 [DOI]

    Data Availability Statement

    Underlying data

    Figshare: Pseudomonas aeruginosa pyocyanin, https://doi.org/10.6084/m9.figshare.13247429.v1 36.

    Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

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