Hypertonic glucose inhibits growth and attenuates virulence factors of multidrug-resistant Pseudomonas aeruginosa by regulating quorum sensing

doi:10.21203/rs.2.21617/v1

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Hypertonic glucose inhibits growth and attenuates virulence factors of multidrug-resistant Pseudomonas aeruginosa by regulating quorum sensing

https://doi.org/10.21203/rs.2.21617/v1

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Background: Pseudomonas aeruginosa is the most common Gram-negative pathogen responsible for chronic wound infections such as diabetic foot infections, which make chronic wound treatment become increasingly difficult and costly, further exacerbated. Hypertonic glucose, a commonly used prolotherapy solution, can accelerate the proliferation of granulation tissue and improve microcirculation in the wound, but the knowledge of the impacts that hypertonic glucose has on wound infecting pathogens has lagged. In this study, we aim to investigate the inhibitory effects of hypertonic glucose on diabetic foot infecting multidrug-resistant P. aeruginosa, to offer a novel approach to control the chronic wound infections caused by P. aeruginosa.

Results: Four multidrug-resistant P. aeruginosa clinical strains isolated from diabetic foot ulcers from a tertiary hospital in China and P. aeruginosa PAO1 were studied. Hypertonic glucose had a remarkable inhibitory effect on growth of PAO1 and multidrug-resistant P. aeruginosa strains. Furthermore, hypertonic glucose significantly inhibited the formation of biofilm, the ability of swimming, and the production of virulence factors including pyocyanin and elastase in all P. aeruginosa strains. We examined the expressions of major quorum sensing related genes (lasI, lasR, rhlI, and rhlR) in P.aeruginosa and found these genes were all downregulated after hypertonic glucose treatment. In infection model of Galleria mellonella larvae, groups inoculated with P. aeruginosa strains which were treated with hypertonic glucose showed no increased survival rate than non-hypertonic glucose-treated groups.

Conclusions: Our research suggested that hypertonic glucose displayed the ability to inhibit growth, biofilm formation and swimming motility as well as attenuate virulence factors of multidrug-resistant P. aeruginosa via regulating quorum sensing system. Further clinical studies combining hypertonic glucose with traditional antibiotics should be considered in chronic wound infections.

Applied & Industrial Microbiology

hypertonic glucose

Pseudomonas aeruginosa

quorum sensing

virulence factors

biofilm

motility

Pseudomonas aeruginosa is an important human opportunistic pathogen that is able to cause life-threatening infections, particularly in immunocompromised patients and it is also the most common Gram-negative pathogen responsible for infections of chronic wounds, such as venous leg ulcers, pressure ulcers, and diabetic foot ulcers [1–3]. Antibiotics have long become the approach in our effort to treat these infectious diseases [4]. However, extensive use of these agents creates an evolutionary pressure on P. aeruginosa which, in many cases, leads to antibiotics resistance, even multidrug resistance. Therefore, rational strategies with low potential for resistance emergence are needed in order to combat pathogenic multidrug-resistant P. aeruginosa.

To the best of our knowledge, hypertonic glucose, the commonly used prolotherapy solution [5], can accelerate the proliferation of granulation tissue and improve microcirculation in the wound, which will reduce the time of healing or further management [6]. Moreover, there was a case report showing that the combination of hypertonic glucose and negative pressure wound therapy was found to be safe and effective in preparing the wound bed with P. aeruginosa infection for grafting [7]. Despite several reports showing hypertonic glucose is effective in the treatment of chronic wound injuries, the understanding of hypertonic glucose against wound bacterial infections has lagged.

It has been reported that numerous factors like biofilm, motility, and virulence factors such as pyocyanin and elastase are required in P. aeruginosa for pathogenicity [8–10]. These traits of P. aeruginosa are regulated by quorum sensing (QS), which monitors bacterial population density and several physiological processes [11, 12]. P. aeruginosa has three QS systems, las, rhl, and pqs [13]. The signal molecules N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), Nbutanoyl-L-homoserine lactone (C4-HSL), and Pseudomonas quinolone signal (PQS) are produced by LasI, RhlI, and PqsA, respectively [2]. Moreover, the corresponding receptors are LasR, RhlR, and PqsR, respectively. Specific autoinducer (AI) binding to its receptor is able to activate genes responsible for pathogenic phenotype of P. aeruginosa [14]. Therefore, utilization of anti-QS strategies is probably a promising way to tackle P. aeruginosa infections.

In this research, we investigated the antimicrobial and anti-quorum sensing potential of hypertonic glucose on P. aeruginosa PAO1 and four multidrug-resistant P. aeruginosa strains separated from diabetic foot ulcers, to offer a new insight in chronic wound infection treatment.

Bacterial strains and antimicrobial susceptibility profiling

Four multidrug-resistant P. aeruginosa strains (TL2941, TL3147, TL3445 and TL3581) clinically isolated from different diabetic foot ulcers from the First Affiliated Hospital of Wenzhou Medical University in China, and P. aeruginosa strain PAO1 were used in this study. Identification and antimicrobial susceptibility testing were conducted on clinical isolates using VITEK MS system and VITEK2 system, respectively. All strains were kept in our laboratory and propagated in LB medium (1% tryptone, 1.0% NaCl, 0.5% yeast extract) at 37 °C.

Determination of minimum inhibitory concentration

The minimum inhibitory concentration (MIC) values of glucose (D-(+)-Glucose, Sigma, St. Louis, USA) were determined as previously published methods, with some modifications [15]. Briefly, bacterial suspension (10⁸ CFU/ mL) of P. aeruginosa PAO1 and multidrug-resistant P. aeruginosa strain were added to LB broth (1%, v/v) with glucose at concentration gradients (0, 50, 100, 150, 200, 250, 300 and 350 mg/mL) in 96-well microtiter plates, then incubated at 37 °C for 18 h. The MIC was the lowest concentration of glucose with visible inhibition of cell growth. The MICs of glucose against all tested P. aeruginosa are recorded in Table 1. All further experiments in this study were conducted at sub-minimum inhibitory concentrations (sub-MICs).

Growth curves asssay

Effects of glucose on the growth of all tested P. aeruginosa strains were determined according to the method described previously, with some modifications [16]. Briefly, the overnight cultures were diluted 1:100 into fresh LB broth for incubation and OD₆₀₀values of subcultures were adjusted to 0.1, then added to the 96-well microtiter plates containing LB with different final concentrations of glucose (0, 50, 100, 150 and 200 mg/mL). LB medium and LB with glucose were used as negative control. The 96-well microtiter plates were incubated at 37 °C. The absorbance of each sample at OD₆₀₀ nm was measured every 1 h.

Biofilm formation inhibition

Crystal violet method was used to test the biofilm forming capacity of all P. aeruginosa. Briefly, all selected strains were grown overnight in LB broth. The overnight cultures were then diluted 1:100 in fresh LB broth supplemented with different glucose concentrations (0, 50, 100, 150 and 200 mg/mL). A total of 100μL of each dilution were added to a 96-well microtiter plate and incubated at 37 ℃ for 24 h. Wells containing media alone were used as blank. Planktonic cells were removed and the wells were washed twice with sterile water, then the wells were stained with 150 μL 0.1% crystal violet for 10 min and rinsed twice with sterile water. Stained biofilms were solubilized with 95% ethanol and quantified by measuring the OD₅₉₅ using a microplate reader.

Swimming motility assay

The swimming assay was conducted as prior published methods, with some modifications [15]. Briefly, the filtered different concentrations of glucose were added to molten swimming agar (pH 7.2), which consisted of 0.1% tryptone, 0.05% yeast extract, 0.5% NaCl, 0.3% Bacteriological agar. The molten agar was then dispensed onto Petri dishes after gentle mixing. Once the agar with glucose (0, 50, 100, 150, 200 and 300 mg/mL) was solidified, 2µL of P. aeruginosa culture was inoculated in the center of the agar and then incubated at 37 ℃ for 24 h. We used swimming agar and swimming agar with 300 mg/mL glucose as the control.

Pyocyanin assay

Pyocyanin production was examined according to a protocol described previously with modification [17]. 1 ml of the bacterial cultures under different concentrations of glucose conditions (0, 50, 100, 150 and 200 mg/mL) were subject to centrifugation at 13,000 rpm for 5 min. The supernatant was collected and extracted with 600 μl chloroform following vortex for 10s twice. After centrifugation at 13,000 rpm for 5 min, the chloroform phase was transferred into a sterile tube, and subsequently mixed with 0.5 ml 0.2 M HCl followed by gentle shaking to transfer the pyocyanin to the aqueous phase. Pyocyanin was determined by measuring the absorbance of the aqueous phase at 510 nm.

Elastase activity assay

The elastolytic activity of the cell-free culture supernatant of P. aeruginosa was determined following the method described previously by using Elastin-Congo red (ECR; Sigma, St. Louis, USA) as the substrate [18]. Then, 100 μL supernatants of P. aeruginosa incubated in different concentrations of glucose (0, 50, 100, 150 and 200 mg/mL) were added to 900 μl of ECR buffer (100mM Tris, 1mM CaCl2, pH 7.5) containing 10 mg of ECR and incubated at 37 °C for 3 h. The reaction was terminated by adding 1ml of 0.7 M sodium phosphate buffer (pH 6.0) and the tubes were placed in cold water bath. The insoluble ECR was removed by centrifugation at 10,000 rpm for 10 min and then the absorbance was measured at OD₄₉₅nm.

Infection model of Galleria mellonella larvae

mellonella killing assays were carried out on the P. aeruginosa as described previously, with some modifications [19]. Nine larvae weighing between 200 mg-250 mg were randomly selected for each strain. The overnight cultures were diluted 1:100 in fresh LB broth supplemented with different concentrations of glucose (0, 100, 150 and 200 mg/mL) and incubated at 37 ℃ for 8h. Then the bacteria were rinsed three times using phosphate-buffered saline (PBS) and resuspended in PBS. A 10 μL of bacterial suspension (10⁷CFU/mL) was injected into the last left proleg using a 25 μL Hamilton precision syringe. Larvae injected with 10 μl PBS were used as control. The insects were incubated at 37 ℃ in the dark and observed after 24 h, 48 h and 72 h. Larvae were considered dead when they repeatedly failed to respond to physical stimuli. The primary outcome for the insect model was rapidity and extent of mortality of G. mellonella assessed with Kaplan-Meier analysis and log-rank test.

Quantitative reverse transcription PCR (qRT-PCR)

The effects of hypertonic glucose on the expression levels of P. aeruginosa major QS circuit genes (lasI, lasR, rhlI, and rhlR) were evaluated using quantitative reverse transcription PCR (qRT-PCR). For RNA extraction, P. aeruginosa isolates were grown in fresh LB medium with or without 200 mg/mL glucose at 37 ℃ for 17 h. Total RNA was extracted using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The extracted RNA samples were stored at −80 ℃. Purified RNA was reverse transcribed into cDNA for qRT-PCR analysis using a cDNA synthesis kit (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. Gene expression levels were measured with qRT-PCR using a 7500 RT-PGE system (TOYOBO, Osaka, Japan) and SYBR Green qRT-PCR Kit (TOYOBO) with the specific primers listed in Table 2. The rpsL gene was used as an internal control to normalize the data. Gene expression levels were calculated using 2^−△△Ct method.

Statistical analysis

All experiments were conducted independently with at least two replicates on different days, and results were expressed as mean ± standard deviation. The total area under the curve was calculated for analysis on growth. T-test was used to evaluate the significance of differences between two groups. One-way analysis of variance (ANOVA) was performed to analyze the significance among more groups. Statistical significance was determined at P < 0.05. Statistical analyses were performed using SPSS version 17.0 statistical software.

Antimicrobial susceptibility profiles and antimicrobial activity of hypertonic glucose

Antibiotic resistance profiles of four multidrug-resistant P. aeruginosa strains were displayed in Table 3. Broth micro-dilution method was used to determine the MICs of glucose against different strains. As presented in Table 1, glucose inhibited all multidrug-resistant P. aeruginosa strains (TL2941, TL3147, TL3445 and TL3581) and P. aeruginosa strain PAO1 at the concentration of 300 mg/mL. In addition, hypertonic glucose showed a concentration-dependent inhibitory effect on the growth of all P. aeruginosa strains (P < 0.05) (Figure 1).

Hypertonic glucose affects biofilm formation and cell swimming motility of P. aeruginosa

The effects of hypertonic glucose on biofilm formation were studied in P. aeruginosa using crystal violet assay. Hypertonic glucose decreased the biofilm forming capacity of all tested P. aeruginosa although the concentration depend tendency was not apparent (P < 0.05) (Figure 2). Furthermore, hypertonic glucose showed a concentration-dependent inhibitory effect on the swimming motility of all selected P. aeruginosa strains (P < 0.05) (Figure 3). At 300 mg/mL, the turbid zone disappeared. The turbid zone spread throughout the whole swimming agar when no glucose existed.

Hypertonic glucose influences virulence factors of P. aeruginosa

The effects of hypertonic glucose on virulence factors such as elastase and pyocyanin were also examined. As shown in Figure 4, hypertonic glucose significantly inhibited the production of elastase and pyocyanin in PAO1 and all multidrug-resistant P. aeruginosa strains at all tested concentrations. The finding presented in Figure 4 showed that 100 mg/mL glucose caused a significant decrease of above 80% in production of pyocyanin and elastase (P < 0.05). Since TL3581 P. aeruginosa strain produced limited elastase, which could not be detected by Elastin-Congo red method, the percentage inhibition rate was not calculated.

Hypertonic glucose regulates the expression of QS-related genes

Despite the inhibitory effects on biofilm formation, swimming motility and the production of virulence factors, effect of hypertonic glucose on QS was not confirmed. Thus, in this study, we evaluated the expressions of QS-regulated genes in P. aeruginosa to elucidate the QS-regulating effect of hypertonic glucose at the transcriptional level. The relative expression of genes lasI, lasR, rhlI, and rhlR in PAO1 and 4 multidrug-resistant P. aeruginosa strains were determined from calculated Ct values. In all tested P. aeruginosa, the QS-related genes were significantly downregulated under hypertonic glucose condition as shown in Figure 5 (P < 0.05).

Hypertonic glucose can not decrease mortality in infection model of G. mellonella larvae

To investigate effect of hypertonic glucose on P. aeruginosa virulence, in vivo infection model G. mellonella were used. All isolates caused a time-dependent death of the larvae. Moreover, for all tested P. aeruginosa strains, there was no significant difference in mortality among groups of different glucose concentrations as Figure 6 showed (P > 0.05).

P. aeruginosa is among the top three major human opportunistic pathogens that cause mortality and morbidity in immunocompromised patients and it is a clinical common pathogen relevant with chronic wound infections such as diabetic foot infections [20]. The presence of P. aeruginosa in chronic leg ulcers can induce ulcer enlargement and/or cause delayed healing, which should be greatly considered clinically [21]. P. aeruginosa has acquired resistance for widespread use of antibiotics and new approaches are urgently needed. The commonly used prolotherapy solution, hypertonic glucose, has capacity to reduce the time of healing or further management in wounds, but the knowledge of how hypertonic glucose influences wound infecting bacteria is limited. To date, this is the first study investigating the effects of hypertonic glucose on the most common wound infecting Gram-negative pathogens P. aeruginosa.

In this study, we demonstrated hypertonic glucose had an antimicrobial activity and showed significant inhibitory effect on the growth of P. aeruginosa PAO1 and 4 multidrug-resistant strains. We further explored its quorum-sensing quenching activity by P. aeruginosa at sub-MICs. The pathogenesis of P. aeruginosa relies largely on motility and the production of virulence factors such as pyocyanin and elastase, which are elaborately controlled by the hierarchal QS systems [22]. In addition, P. aeruginosa frequently forms biofilms on medical surfaces, and biofilms are prevalent in chronic wounds and once formed are very hard to remove, which is associated with poor outcomes [23, 24]. In P. aeruginosa, hypertonic glucose significantly inhibited the formation of biofilm, the ability of swimming, and the production of virulence factors including elastase and pyocyanin.

Targeting QS becomes an attractive option for infection treatment, which could minimize selection pressures and the risk of drug resistance. In the las and rhl systems, the virulence factors of P. aeruginosa are primarily encoded by QS-regulated genes lasI, lasR, rhlI, and rhlR [22]. It has been known that LasR and RhlR interacting with 3-oxo-C12-HSL and C4-HSL, respectively, can trigger the production of pyocyanin and elastase in P. aeruginosa [25]. In this work, we investigated whether the expressions of major QS-regulated genes lasI, lasR, rhlI, and rhlR could be influenced by hypertonic glucose to attenuate P. aeruginosa virulence factors. As expected, average relative amounts of QS-regulated genes were normalized to the average relative amount of the rpsL reference gene, with lasI, lasR, rhlI,and rhlR found to be significantly decreased by hypertonic glucose (P < 0.05). It is possible that hypertonic glucose creates a harsh environment, making P. aeruginosa incapable of increased expression of QS-regulated genes to keep the production of virulence factors. Thus, in this study, the significantly decreased expression of QS-regulated genes resulted in the decrease in production of virulence factors and swimming motility. Our results indicated that hypertonic glucose could attenuate P. aeruginosa virulence factors via regulation of the QS system.

We used G. mellonella infection model to explore the anti-virulence ability of hypertonic glucose in vivo. It is intriguing to note that there was no significant difference in mortality among groups of different glucose concentrations (P > 0.05). The results were probably attributed to P. aeruginosa could produce virulence factors rapidly and restore virulence once it get rid of the hypertonic glucose environment.

It is well recognized that QS plays an important role in the wound infection [26] and chronic wound treatment is becoming increasingly difficult and costly, further exacerbated when wounds become infected. The results strongly suggested hypertonic glucose could be used as a promising agent for chronic wound infections, since hypertonic glucose, as a commonly used prolotherapy solution, showed in vitro inhibitory effect of growth and attenuated virulence factors via regulation of the QS system. However, because of the limited laboratory conditions, mouse wound infection model and clinical trials were not performed to demonstrate the hypothesis.

The exploitation of QS system to search for virulence inhibitors has offered an alternative opportunity to control bacterial infections, given the fact that traditional antimicrobial treatment could easily develop resistance to antibiotics.The combination of the drugs with QS inhibitor will greatly impair bacterial infections, especially for the control of biofilm-related issues [27, 28]. In this study, hypertonic glucose could inhibit growth and attenuate virulence factors of multidrug-resistant P. aeruginosa by regulating quorum sensing without developing resistance to antibiotics. Based on this notion, further combinatory measurement including hypertonic glucose with traditional antibiotics should be considered to enhance the therapeutic effect of bacterial infections.

In conclusion, to the best of our knowledge, this is the first study to report on the inhibitory effects of hypertonic glucose on a common wound infecting species, P. aeruginosa. Hypertonic glucose displayed the ability to inhibit growth, biofilm formation and swimming motility as well as attenuate virulence factors of multidrug-resistant P. aeruginosa via regulating QS system. Pending clinical studies combining hypertonic glucose with traditional antibiotics should be considered in wound infections.

Ethics approval and consent to participate

Ethics approval and consent is deemed unnecessary in this research according to the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and analysed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

The work was financially supported by the grant from the Zhejiang Province Natural Science Foundation of China (No. LY18H190004) and the Health Department of Zhejiang Province of the People's Republic of China (No. 2018KY123).

Author Contributions

All authors contributed conception and design of the study; TC, YX, WX ,WL, CX and XZ acquired the data; TC performed the statistical analysis; TC wrote the first draft of the manuscript; JC and TZ supervised the study. All authors contributed to manuscript revision, read and approved the submitted version.

Acknowledgments

The authors acknowledge the financial support from the Zhejiang Province Natural Science Foundation of China (No. LY18H190004) and the Health Department of Zhejiang Province of the People's Republic of China (No. 2018KY123).

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	Strains	Glucose MIC values (mg/ml)
MDR clinical isolates	TL2941	300
	TL3147	300
	TL3445	300
	TL3581	300
Reference isolates	PAO1	300

Table 1. Minimum inhibitory concentrations of glucose against MDR Pseudomonas aeruginosa clinical isolates and PAO1.

Gene Name	Type	Primer sequence	Annealing temp (°C)	Amplicon size (bp)
lasI	F	5‘- GGCTGGGACGTTAGTGTCAT-3’	58	104
	R	5‘-AAAACCTGGGCTTCAGGAGT-3’
lasR	F	5‘-ACGCTCAAGTGGAAAATTGG-3’	58	111
	R	5‘-TCGTAGTCCTGGCTGTCCTT-3’
rhlI	F	5‘-AAGGACGTCTTCGCCTACCT-3’	58	130
	R	5‘- GCAGGCTGGACCAGAATATC-3’
rhlR	F	5‘-CATCCGATGCTGATGTCCAACC-3’	60	101
	R	5‘-ATGATGGCGATTTCCCCGGAAC-3’
rpsL	F	5‘-GCAACTATCAACCAGCTGGTG-3’	58	231
	R	5‘-GCTGTGCTCTTGCAGGTTGTG-3’

Table 2. Primers used in qRT-PCR for quorum sensing circuit genes lasI, lasR, rhlI, and rhlR, and reference gene, rpsL.

Strains	Antimicrobial resistance
Strains	AMK	TOB	GEN	CAZ	FEP	ATM	IPM	TZP	SCF	CIP	LVX
TL2941	S	R	R	I	I	R	I	R	I	R	R
TL3147	R	R	R	S	R	R	S	R	I	R	I
TL3445	S	R	R	R	R	N	R	I	S	R	R
TL3581	R	R	R	S	R	N	S	R	I	R	R

Table

Table 3. Summary of antibiotic resistance profiles of multidrug-resistant Pseudomonas aeruginosa.

AMK, Amikacin; TOB, Tobramycin; GEN, Gentamicin; CAZ, ceftazidime; FEP, Cefepime; ATM, Aztreonam; IPM, imipenem; TZP, Piperacillin-tazobactam; SCF, Cefoperazone-sulbactam; CIP, ciprofloxacin; LVX, Levofloxacin; S, susceptible; R, resistant; I, intermediate; N, Not done.

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Hypertonic glucose inhibits growth and attenuates virulence factors of multidrug-resistant Pseudomonas aeruginosa by regulating quorum sensing

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