Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa [version 1; peer review: 1 approved with reservations, 1 not approved]

Background: Pseudomonas aeruginosa, a multidrug resistant Gramnegative 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® 10145TM 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® 10145TM, 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 Open Peer Review


Introduction
Pseudomonas aeruginosa (P. aeruginosa) is one of the Gramnegative bacilli bacteria which causes nosocomial infections that can be fatal, especially in immunocompromised patients [1][2][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][6][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][16][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 pyocyaninproducer 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 .

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%.
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.
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.
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.

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

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.

Author Response 07 Jun 2021
Heni Susilowati, Faculty of Dentistry, Universitas Gadjah Mada, Sleman, Indonesia 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

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:

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 yesthen 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.
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 yesthen 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.

Are the conclusions drawn adequately supported by the results? Yes
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