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Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem
JBUR-4749; No. of Pages 6 burns xxx (2016) xxx–xxx
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/burns
Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem Soliman Mohammadi-Samani a,*, Shahriyar Kouroshfard b, Negar Azarpira c a
Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Shiraz University of Medical Science, Shiraz, Iran Faculty of Paramedical Sciences, Shiraz University of Medical Science, Shiraz, Iran c Shiraz Transplant Research Center, Shiraz University of Medical Science, Shiraz, Iran b
article info
abstract
Article history:
This study was designed to investigate the effect of inorganic phosphate supplementation on
Accepted 3 September 2015
invasive behavior of Pseudomonas aeruginosa in burn wound infections. An emulsion-based lotion containing sodium dihydrogen phosphate was formulated and then 50 female Sprague-
Keywords:
Dawley rats with burn wounds were used to assess the effect of phosphate supplementation
Burn
on swarming motility of P. aeruginosa. On the second day after burn, four groups of rats were
Microbial resistance
inoculated with P. aeruginosa and one group was left as negative control. The treatment was
Phosphate
started on day 3 and the animals were followed up for 4 weeks. Significant improvement in
Pseudomonas
wound healing was observed in the phosphate-receiving group after the 4-week follow-up, compared to the negative control, positive control, and silver sulfadiazine-receiving groups. Histopathological assessment of the tissue samples also indicated the healing process in phosphate-enriched lotion receiving group. The results showed that inorganic phosphate supplementation results in alteration of the virulence behavior of P. aeruginosa and improvement in the wound healing process. In conclusion, phosphate supplementation would be a rational strategy in the eradication of P. aeruginosa wound infection. # 2015 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Skin burn is one of the most common forms of trauma. Although critical care of burn-related trauma has improved considerably during the last decades, there are still many reports confirming the mortality rate in patients with burns over more than 40% of the total body surface area [1,2].
Bacterial superinfection of the burn wounds, especially with Pseudomonas aeruginosa, is a common complication in patients with burn trauma [1]. A number of protocols are available for local and systemic treatment to minimize superinfection. Nevertheless, severe burn wounds are infected at relatively high rates with various environmental and nosocomial bacteria. P. aeruginosa accounts for approximately half of all severe burn infections [2]. Biofilm formation by these bacteria
* Corresponding author at: Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Shiraz University of Medical Science, Shiraz, Iran. Tel.: +98 7132426070; fax: +98 7132424126. E-mail address: [email protected] (S. Mohammadi-Samani). http://dx.doi.org/10.1016/j.burns.2015.09.003 0305-4179/# 2015 Elsevier Ltd and ISBI. All rights reserved.
Please cite this article in press as: Mohammadi-Samani S, et al. Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem. Burns (2016), http://dx.doi.org/10.1016/j.burns.2015.09.003
JBUR-4749; No. of Pages 6
2
burns xxx (2016) xxx–xxx
provides survival advantages and resistance to host immune responses or antibiotics [3,4]. P. aeruginosa is a Gram-negative opportunistic pathogenic bacterium which is normally found in water, soil, and vegetation [5]. Bacterial colonization and the subsequent infection in patients occur in several conditions such as trauma, surgery, or insertion of various devices such as urinary catheter or electrode. The bacterium can also colonize through disruption of the normal flora balance during the administration of broad-spectrum antibiotics or immune system dysfunction [6]. P. aeruginosa causes life-threatening infections in various tissues and organs and despite the availability of various antibiotics during the last decades, the eradication of these infections is still difficult [7]. Wound superinfection due to bacterial contamination is still considered as a common complication in patients with burns, potentially leading to considerable morbidity and mortality [8– 10]. Based on the literature, sepsis accounts for 50–60% of mortality in patients with burns despite improvements in antimicrobial therapies [11,12]. The antibiotic treatment of P. aeruginosa infections may actually cause drug resistance and survival of selected pathogenic variants [13,14]. Studies conducted in some experimental models confirm that phosphate depletion induces phenotypic changes in P. aeruginosa [15]. It has been proposed that phosphate depletion in host organs induces three global virulence pathways in P. aeruginosa. They include phosphate signaling (phosB), the MvfR-PQS pathway of quorum sensing, and the pyoverdinmediated iron acquisition system [15]. In addition, some reports indicate injury-induced phosphate depletion [16]. The present study aims to determine whether inorganic phosphate supplementation prevents the activation of lethal phenotype in P. aeruginosa in a rat model.
2.
Materials and methods
2.1.
Materials
Ketamine, xylazine, vaseline, cetostearyl alcohol, sodium dihydrogen phosphate, glycerin, and liquid paraffin were purchased from Merck (Germany). Methyl hydroxybenzoate and propyl hydroxybenzoate were procured from Sigma (USA) and cetomacrogol 1000 was from Darou Pakhsh, Iran. Silver sulfadiazine ointment was purchased from the domestic market in Shiraz, Iran. All other chemicals and reagents were of analytical grade and were used without any modification.
2.2.
Methods
2.2.1.
Lotion formulation
Sodium dihydrogen phosphate, glycerin, methyl hydroxybenzoate and propyl hydroxybenzoate were mixed at appropriate concentrations with distilled water and heated up to 75 8C in a beaker. Vaseline, liquid paraffin, cetostearyl alcohol and cetomacrogol 1000 were heated up to 75 8C in a separate beaker. Then, the beakers were removed from the water bath, and the aqueous phase was added to the oil phase followed by mixing at 500 rpm for 10 min using a drive mixer. Once the lotion was cooled, it was packed in an appropriate jar and kept
in refrigerator for use in animal study. Sodium dihydrogen phosphate concentration was 80 mM in phosphate-enriched formulation.
2.2.2.
Particle size and Zeta potential determination
Particle size and Zeta potential of the base emulsion and phosphate-enriched emulsion were determined using Microtrac instrument (Germany). In this case, 1 g of emulsion was diluted in 100 ml of distilled water and 1 ml of the diluted samples was used to measure the Zeta potential and particle size. The mean volume based size of the emulsified phase was 977 nm in base formulation and 684 nm in phosphate-enriched formulation, respectively. The mean Zeta potential of the emulsified oil was 97.6 1.2 mV for base formulation and 70.2 0.4 mV for phosphate-enriched formulation, respectively.
2.2.3.
Ethic statement
Animal studies were conducted according to the approved protocols of Shiraz University of Medical Sciences (Shiraz, Iran) for animal handling. The ethical code of the animal study was ec_p_91_4490 and was approved on 1/6/2013 by the Ethics Committee of Shiraz University of Medical Sciences.
2.2.4.
Animal study
A total of 50 female Sprague Dawley rats (230 50 g), aged between 8 and 10 weeks, from the Laboratory Animal Center of Shiraz University of Medical Sciences, Shiraz, Iran were randomly divided into five different groups. All the rats were anesthetized using ketamine–xylazine cocktail (100 mg/kg ketamine and 10 mg/kg xylazine). Then, the back of the anesthetized rats was shaved with scissors and the predetermined sites were burnt for the same duration using a hot square plate of 1-cm2 surface area, which had been heated on top of the flame. P. aeruginosa was isolated from the infected burn wound of the patient in Ghotbedin Burn Hospital, Shiraz, Iran. Preliminary identification of isolated bacteria was performed by colony morphology, odor, zone of hemolysis and oxidase, methyl red, Voges–Proskauer, and citrate tests and finally the isolated strain PTCC number (1811) was specified by Iranian Research Organization for Science and Technology (IROST). This strain was resistant to gentamicin, tetracycline, tobramycin, ceftazidime, ceftriaxone, ciprofloxacin, carbenicillin, imipenem, and intermediate to piperacillin–tazobactam. P. aeruginosa was cultured and incubated for 24 h. It was then suspended in normal saline solution and the microbial concentration was adjusted to 1.2 109 CFU/ml (4 Macfarlane). P. aeruginosa suspension of 1.2 109 CFU/ml was applied to the inflamed areas of four rat groups and one group was assigned as the negative control group (group 2). Each rat was kept in isolated separate cage to prevent the transmission of infection between animals. At 24 h after inoculation and confirmation of the infection in the injured areas, treatment was started, according to the following group classification: Phosphate-receiving group: Includes 10 infected rats. Phosphate-enriched lotion formulation was applied on the surface of the wounds one time a day. Negative control group: Includes 10 rats that were burnt. P. aeruginosa suspension was not applied, neither did they receive any treatment.
Please cite this article in press as: Mohammadi-Samani S, et al. Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem. Burns (2016), http://dx.doi.org/10.1016/j.burns.2015.09.003
JBUR-4749; No. of Pages 6 burns xxx (2016) xxx–xxx
Positive control group: Includes 10 rats that were burnt. P. aeruginosa suspension was applied, but they did not receive any treatment. Silver-receiving group: Includes 10 rats that were burnt. P. aeruginosa, suspension was applied and they received standard treatment with silver sulfadiazine ointment. Base-receiving group: Includes 10 rats that received only the lotion base with no source of phosphate to evaluate the healing effect of lotion base. Clinical evaluation of the wounds including wound area size and crust formation was performed at the end of each week for 4 weeks.
2.2.5.
Microbiological and histopathological evaluation
On the 28th day, the animals were anesthetized and then decapitated. Sterile culture from wound area was taken to the Microbiology Department immediately and seeded into several plates (thioglycollate, blood agar, chocolate agar, and MacConkey agar) to maximize the number of bacterial species that could be isolated. Organisms were identified using Gram staining, catalase and coagulase, eosin methylene blue (EMB), triple sugar iron agar (TSI), sulfide indole motility (SIM), urea broth, Simmons citrate and methyl red, and Voges– Proskauer broth (MR-VP) test. The wounded skin was carefully excised for histopathological examination. It was fixed in 10% buffered formalin solution and embedded in paraffin blocks. Sections were stained using hematoxylin and eosin method (H&E). Skin tissue sections (5 mm) were prepared and examined under a light microscope (Olympus, Japan). Histopathological evaluation was done by an experienced histopathologist who was blind to treatment conditions. Grading of the findings was done by assigning scores, 0: none; 1: mild; 2: moderate, and 3:
3
severe for each of the following criteria: surface ulceration, reepithelialization, granulation tissue, and existence of neutrophil and leukocyte infiltration (sign of acute inflammation) and monocytes/plasma cell (sign of chronic inflammation). Reepithelialization refers to complete epithelialization or wound healing (grade 3), in the absence of ulceration. Any defect in the wound surface indicates that healing was incomplete (grade 1 or 2) and no epithelialization is assigned grade zero [17]. At least five microscopic fields were evaluated to score each specimen.
2.3.
Statistical analysis
Statistical analysis was carried out using SPSS (version 20) Software, USA. All data are expressed as means SD. The obtained data were compared with analysis of variance (ANOVA) technique, followed by Tukey’s comparison tests. P < 0.05 was regarded as statistically significant
3.
Results
Each group consisted of 10 rats. In positive control and silverreceiving groups, three rats died. In base-receiving group, seven animals died during the follow-up period. In microbiological studies, the presence of both gram-positive and gramnegative bacteria such as Klebsiella, Streptococcus, Helicobacter, Citrobacter, and Staphylococcus was confirmed. P. aeruginosa was isolated from all groups except the phosphate-receiving group. Average diameter of the wounds at the beginning of study (day 1) was 1.7 0.2 cm. All of the rats in different groups were controlled during the 28 days of the experiment and the wound healing rate was measured after this period (Fig. 1). In
Fig. 1 – Mean size of the wounds in different groups of the treatment. Please cite this article in press as: Mohammadi-Samani S, et al. Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem. Burns (2016), http://dx.doi.org/10.1016/j.burns.2015.09.003
JBUR-4749; No. of Pages 6
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burns xxx (2016) xxx–xxx
Fig. 2 – Histopathological representation of the wounds after 28 days after treatment protocols (H&E 400T); reepithelialization is shown as green arrow. The surface ulceration is marked as yellow arrow. Complete surface ulceration with no re-epithelialization is noticed in burned skin infected with bacteria (positive control), in silver sulfadiazine receiving group, and in lotion base receiving group. Partial re-epithelialization is observed in negative control group in which burned skin was left without any treatment (positive control). Complete surface re-epithelialization with no evidence of surface ulceration was noticed in phosphate enriched lotion receiving group. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)
phosphate-enriched lotion-receiving group, there was significant improvement in wound healing (0.06 0.09 cm), compared to the other groups (P < 0.05). In this group, almost all the ulcers were fully healed, but in groups 3, 4, and 5, there were large open wounds. The wound diameter in the base-receiving group increased (1.9 0.1 cm), but in the positive control and silver-receiving groups that received standard sulfadiazine treatment or were left untreated, no significant increase was observed (P > 0.05) (Fig. 1).
Based on the results given in Fig. 2, microscopic examination of the wound samples revealed that surface ulceration significantly decreased in the phosphate-receiving group, in comparison to that in other groups (P < 0.05). Besides, as shown in Fig. 2, the reepithelialization and granulation tissue formation were significantly better in the phosphate-receiving group, compared to others (P < 0.05). The acute inflammatory cell infiltration decreased in the phosphate-receiving group (P < 0.05), but there was no significant difference in chronic inflammation across the groups (Table 1).
Table 1 – Pathologic score of wound healing in different treatment groups. Surface ulceration G1 G1 G1 G1
vs vs vs vs
G2 G3 G4 G5
15.19 vs 13.58** 9.06 vs 21.75* 9.34 vs 24.75* 11.50 vs 24.10*
Re-epithelialization 13.22 19.84 24.47 25.78
vs vs vs vs
16.21** 7.38* 11.31* 12.68*
Granulation tissue formation 17.25 12.41 15.22 17.94
vs vs vs vs
10.83** 17.29** 18.68** 18.95**
Acute inflammation 16.47 11.53 13.75 14.94
vs vs vs vs
11.88** 18.46* 20.83* 21.35**
Chronic inflammation 16.19 14.59 18.09 21.97
vs vs vs vs
12.25** 14.38** 15.97** 15.73**
Note: All groups were compared with G1. G1: phosphate lotion receiving group; G2: negative control group; G3: positive control group; G4: silver sulfadiazine receiving group; and G5: base lotion receiving group. * Significant (P-value < 0.05). ** Not significant (P-value > 0.05).
Please cite this article in press as: Mohammadi-Samani S, et al. Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem. Burns (2016), http://dx.doi.org/10.1016/j.burns.2015.09.003
JBUR-4749; No. of Pages 6 burns xxx (2016) xxx–xxx
4.
Discussion
The size and Zeta potential of the emulsified phase in phosphate-enriched formulation were less than that of base formulation due to the effect of phosphate ions on modulation of the positive ion intensity in the diffusion layer. In this regard, the net effect of the presence of phosphate ions was a decrease in Zeta potential. In wound healing process, the damaged tissues are restored to their normal condition via several mechanisms including wound contraction that help shrinkage of the wound area and its rate depends on the ability of the tissue to repair. Silver sulfadiazine ointment proved to be an effective antibacterial agent but was shown to delay the healing process in burn wounds due to inhibition of proliferation of the keratinocytes and fibroblast, leading to impairment of the wound healing process [18–20]. During this study, an invasive strain of P. aeruginosa was isolated from the infected burn wound of a patient in Ghotbedin Burn Hospital, Shiraz, southern Iran. An antibiogram test of this strain (PTCC 1811) revealed that it is a multidrug-resistant strain and according to the results, group 4 that received silver sulfadiazine ointment showed no improvement in treatment and P. aeruginosa was not eradicated from the wounds which were more serious than those in positive control group (group 3). On the other hand, group 1, exhibited significant reduction in the wound surface area (P < 0.05) and skin contraction was almost complete. The wounds of the rats in the negative control group were in a better condition than in the positive control group. Phosphate is an essential chemical entity for all living organisms. Phosphate participates in critical biochemical processes that are essential for maintaining energy dynamics in the living cells and is also a principal component of nucleic acids and phospholipids in the cell membrane. Phosphate depletion during major traumatic conditions such as surgery and burn has been reported previously [16]. Exposure of P. aeruginosa to subinhibitory antibiotic concentrations and nutrient-deficient environment leads to remarkable changes in gene expression and alteration in virulence and resistance of this bacterium to antibiotics [21–23]. Gene activation for uptake of inorganic phosphate as well as phosphate acquisition from alternative sources of organic phosphate compensates for phosphate limitation for P. aeruginosa and the consequence of this process is probably change in virulence behavior and swarming motility. Previous studies also confirm huge transcriptional changes in genes related to phosphate acquisition, quorum sensing, chemotaxis, toxin secretion, and regulation when P. aeruginosa is grown in a low-phosphate medium [23]. In this regard, we aimed to supply sufficient quantity of inorganic phosphate to upregulate the expression of different genes involved in phosphate acquisition process. The consequences of this process seemed to change in virulence and swarming behavior of P. aeruginosa. According to the results of this study, phosphate supplementation in an adequate concentration probably triggers transcriptional changes in gene regulation to convert the pathogenic and invasive strain of P. aeruginosa to the noninvasive phenotype. We suggest the use of this strategy as a supportive treatment
5
of P. aeruginosa wound infection. In doing so, the need for antibiotic therapy is lessened and would be unnecessary, but further studies are recommended to well establish the usefulness of this new strategy in clinical practices.
Conflict of interest The authors declare that there are no conflicts of interest to disclose.
Acknowledgments The authors acknowledge the financial support of the Shiraz University of Medical Sciences (grant No. 36-4460). The authors wish to thank Hassan Khajehei, PhD, for linguistic editing of the manuscript. The authors also thank Ali Poustforoushfard, Katayoun Javidnia, Mehrdad Vosoughi, Mohammad Taheri, Omid Kouhi, and Hamid Reza Toutounchi for providing technical support.
references
[1] Nichols D, Caceres S, Caverly L, Fratelli C, Kim S, Malcolm K, et al. Effects of azithromycin in Pseudomonas aeruginosa burn wound infection. J Surg Res 2013;183:767–76. [2] Hodle AE, Richter KP, Thompson RM. Infection control practices in US burn units. J Burn Care Res 2006;27:142–51. [3] Klausen M, Heydorn A, Ragas P, Lambertsen L, AaesJørgensen A, Molin S, et al. Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol 2003;48:1511–24. [4] Friedman L, Kolter R. Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms. Mol Microbiol 2004;51:675–90. [5] Rusin PA, Rose JB, Haas CN, Gerba CP. Risk assessment of opportunistic bacterial pathogens in drinking water. Reviews of environmental contamination and toxicology. Springer; 1997: 57–83. [6] Abdelghany SM, Quinn DJ, Ingram RJ, Gilmore BF, Donnelly RF, Taggart CC, et al. Gentamicin-loaded nanoparticles show improved antimicrobial effects towards Pseudomonas aeruginosa infection. Int J Nanomed 2012;7:4053. [7] Krylov V, Shaburova O, Krylov S, Pleteneva E. A genetic approach to the development of new therapeutic phages to fight Pseudomonas aeruginosa in wound infections. Viruses 2013;5:15–53. [8] Abboud MM, Saeed HA, Tarawneh KA, Khleifat KM, Al Tarawneh A. Copper uptake by Pseudomonas aeruginosa isolated from infected burn patients. Curr Microbiol 2009;59:282–7. [9] Capoor MR, Gupta S, Sarabahi S, Mishra A, Tiwari VK, Aggarwal P. Epidemiological and clinico-mycological profile of fungal wound infection from largest burn centre in Asia. Mycoses 2012;55:181–8. [10] Fu Y, Xie B, Ben D, Lv K, Zhu S, Lu W, et al. Pathogenic alteration in severe burn wounds. Burns 2012;38:90–4. [11] Hamrahi V, Hamblin MR, Jung W, Benjamin JB, Paul KW, Fischman AJ, et al. Gram-negative bacterial infection in thigh abscess can migrate to distant burn depending on burn depth. Interdiscip Perspect Infect Dis 2012;2012. [12] Hashimoto MC, Prates RA, Kato IT, Nunez SC, Courrol LC, Ribeiro MS. Antimicrobial photodynamic therapy on
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drug-resistant Pseudomonas aeruginosa-induced infection. An in vivo study. Photochem Photobiol 2012;88: 590–5. Alcala´-Franco B, Montanari S, Cigana C, Bertoni G, Oliver A, Bragonzi A. Antibiotic pressure compensates the biological cost associated with Pseudomonas aeruginosa hypermutable phenotypes in vitro and in a murine model of chronic airways infection. J Antimicrob Chemother 2012;dkr587. Oliver A, Canto´n R, Campo P, Baquero F, Bla´zquez J. High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 2000;288:1251–3. Zaborin A, Romanowski K, Gerdes S, Holbrook C, Lepine F, Long J, et al. Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1. Proc Natl Acad Sci 2009;106:6327–32. Long J, Zaborina O, Holbrook C, Zaborin A, Alverdy J. Depletion of intestinal phosphate after operative injury activates the virulence of P. aeruginosa causing lethal gutderived sepsis. Surgery 2008;144:189–97. Mikus D, Sikiric P, Seiwerth S, Petricevic A, Aralica G, Druzijancic N, et al. Pentadecapeptide BPC 157 cream
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improves burn-wound healing and attenuates burn-gastric lesions in mice. Burns 2001;27:817–27. Atiyeh BS, Costagliola M, Hayek SN, Dibo SA. Effect of silver on burn wound infection control and healing: review of the literature. Burns 2007;33:139–48. Fuller FW. The side effects of silver sulfadiazine. J Burn Care Res 2009;30:464–70. Rashaan ZM, Krijnen P, Klamer RR, Schipper IB, Dekkers OM, Breederveld RS. Non-silver treatment versus silver sulfadiazine in treatment of partial thickness burn wounds in children: a systematic review and meta-analysis. Wound Repair Regen 2014. Breidenstein E, de la Fuente-Nu´n˜ez C, Hancock RE. Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol 2011;19:419–26. Ferna´ndez L, Breidenstein E, Hancock RE. Creeping baselines and adaptive resistance to antibiotics. Drug Resist Updat 2011;14:1–21. Bains M, Ferna´ndez L, Hancock RE. Phosphate starvation promotes swarming motility and cytotoxicity in Pseudomonas aeruginosa. Appl Environ Microbiol 2012;78:6762–8.
Please cite this article in press as: Mohammadi-Samani S, et al. Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem. Burns (2016), http://dx.doi.org/10.1016/j.burns.2015.09.003
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/burns
Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem Soliman Mohammadi-Samani a,*, Shahriyar Kouroshfard b, Negar Azarpira c a
Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Shiraz University of Medical Science, Shiraz, Iran Faculty of Paramedical Sciences, Shiraz University of Medical Science, Shiraz, Iran c Shiraz Transplant Research Center, Shiraz University of Medical Science, Shiraz, Iran b
article info
abstract
Article history:
This study was designed to investigate the effect of inorganic phosphate supplementation on
Accepted 3 September 2015
invasive behavior of Pseudomonas aeruginosa in burn wound infections. An emulsion-based lotion containing sodium dihydrogen phosphate was formulated and then 50 female Sprague-
Keywords:
Dawley rats with burn wounds were used to assess the effect of phosphate supplementation
Burn
on swarming motility of P. aeruginosa. On the second day after burn, four groups of rats were
Microbial resistance
inoculated with P. aeruginosa and one group was left as negative control. The treatment was
Phosphate
started on day 3 and the animals were followed up for 4 weeks. Significant improvement in
Pseudomonas
wound healing was observed in the phosphate-receiving group after the 4-week follow-up, compared to the negative control, positive control, and silver sulfadiazine-receiving groups. Histopathological assessment of the tissue samples also indicated the healing process in phosphate-enriched lotion receiving group. The results showed that inorganic phosphate supplementation results in alteration of the virulence behavior of P. aeruginosa and improvement in the wound healing process. In conclusion, phosphate supplementation would be a rational strategy in the eradication of P. aeruginosa wound infection. # 2015 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Skin burn is one of the most common forms of trauma. Although critical care of burn-related trauma has improved considerably during the last decades, there are still many reports confirming the mortality rate in patients with burns over more than 40% of the total body surface area [1,2].
Bacterial superinfection of the burn wounds, especially with Pseudomonas aeruginosa, is a common complication in patients with burn trauma [1]. A number of protocols are available for local and systemic treatment to minimize superinfection. Nevertheless, severe burn wounds are infected at relatively high rates with various environmental and nosocomial bacteria. P. aeruginosa accounts for approximately half of all severe burn infections [2]. Biofilm formation by these bacteria
* Corresponding author at: Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Shiraz University of Medical Science, Shiraz, Iran. Tel.: +98 7132426070; fax: +98 7132424126. E-mail address: [email protected] (S. Mohammadi-Samani). http://dx.doi.org/10.1016/j.burns.2015.09.003 0305-4179/# 2015 Elsevier Ltd and ISBI. All rights reserved.
Please cite this article in press as: Mohammadi-Samani S, et al. Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem. Burns (2016), http://dx.doi.org/10.1016/j.burns.2015.09.003
JBUR-4749; No. of Pages 6
2
burns xxx (2016) xxx–xxx
provides survival advantages and resistance to host immune responses or antibiotics [3,4]. P. aeruginosa is a Gram-negative opportunistic pathogenic bacterium which is normally found in water, soil, and vegetation [5]. Bacterial colonization and the subsequent infection in patients occur in several conditions such as trauma, surgery, or insertion of various devices such as urinary catheter or electrode. The bacterium can also colonize through disruption of the normal flora balance during the administration of broad-spectrum antibiotics or immune system dysfunction [6]. P. aeruginosa causes life-threatening infections in various tissues and organs and despite the availability of various antibiotics during the last decades, the eradication of these infections is still difficult [7]. Wound superinfection due to bacterial contamination is still considered as a common complication in patients with burns, potentially leading to considerable morbidity and mortality [8– 10]. Based on the literature, sepsis accounts for 50–60% of mortality in patients with burns despite improvements in antimicrobial therapies [11,12]. The antibiotic treatment of P. aeruginosa infections may actually cause drug resistance and survival of selected pathogenic variants [13,14]. Studies conducted in some experimental models confirm that phosphate depletion induces phenotypic changes in P. aeruginosa [15]. It has been proposed that phosphate depletion in host organs induces three global virulence pathways in P. aeruginosa. They include phosphate signaling (phosB), the MvfR-PQS pathway of quorum sensing, and the pyoverdinmediated iron acquisition system [15]. In addition, some reports indicate injury-induced phosphate depletion [16]. The present study aims to determine whether inorganic phosphate supplementation prevents the activation of lethal phenotype in P. aeruginosa in a rat model.
2.
Materials and methods
2.1.
Materials
Ketamine, xylazine, vaseline, cetostearyl alcohol, sodium dihydrogen phosphate, glycerin, and liquid paraffin were purchased from Merck (Germany). Methyl hydroxybenzoate and propyl hydroxybenzoate were procured from Sigma (USA) and cetomacrogol 1000 was from Darou Pakhsh, Iran. Silver sulfadiazine ointment was purchased from the domestic market in Shiraz, Iran. All other chemicals and reagents were of analytical grade and were used without any modification.
2.2.
Methods
2.2.1.
Lotion formulation
Sodium dihydrogen phosphate, glycerin, methyl hydroxybenzoate and propyl hydroxybenzoate were mixed at appropriate concentrations with distilled water and heated up to 75 8C in a beaker. Vaseline, liquid paraffin, cetostearyl alcohol and cetomacrogol 1000 were heated up to 75 8C in a separate beaker. Then, the beakers were removed from the water bath, and the aqueous phase was added to the oil phase followed by mixing at 500 rpm for 10 min using a drive mixer. Once the lotion was cooled, it was packed in an appropriate jar and kept
in refrigerator for use in animal study. Sodium dihydrogen phosphate concentration was 80 mM in phosphate-enriched formulation.
2.2.2.
Particle size and Zeta potential determination
Particle size and Zeta potential of the base emulsion and phosphate-enriched emulsion were determined using Microtrac instrument (Germany). In this case, 1 g of emulsion was diluted in 100 ml of distilled water and 1 ml of the diluted samples was used to measure the Zeta potential and particle size. The mean volume based size of the emulsified phase was 977 nm in base formulation and 684 nm in phosphate-enriched formulation, respectively. The mean Zeta potential of the emulsified oil was 97.6 1.2 mV for base formulation and 70.2 0.4 mV for phosphate-enriched formulation, respectively.
2.2.3.
Ethic statement
Animal studies were conducted according to the approved protocols of Shiraz University of Medical Sciences (Shiraz, Iran) for animal handling. The ethical code of the animal study was ec_p_91_4490 and was approved on 1/6/2013 by the Ethics Committee of Shiraz University of Medical Sciences.
2.2.4.
Animal study
A total of 50 female Sprague Dawley rats (230 50 g), aged between 8 and 10 weeks, from the Laboratory Animal Center of Shiraz University of Medical Sciences, Shiraz, Iran were randomly divided into five different groups. All the rats were anesthetized using ketamine–xylazine cocktail (100 mg/kg ketamine and 10 mg/kg xylazine). Then, the back of the anesthetized rats was shaved with scissors and the predetermined sites were burnt for the same duration using a hot square plate of 1-cm2 surface area, which had been heated on top of the flame. P. aeruginosa was isolated from the infected burn wound of the patient in Ghotbedin Burn Hospital, Shiraz, Iran. Preliminary identification of isolated bacteria was performed by colony morphology, odor, zone of hemolysis and oxidase, methyl red, Voges–Proskauer, and citrate tests and finally the isolated strain PTCC number (1811) was specified by Iranian Research Organization for Science and Technology (IROST). This strain was resistant to gentamicin, tetracycline, tobramycin, ceftazidime, ceftriaxone, ciprofloxacin, carbenicillin, imipenem, and intermediate to piperacillin–tazobactam. P. aeruginosa was cultured and incubated for 24 h. It was then suspended in normal saline solution and the microbial concentration was adjusted to 1.2 109 CFU/ml (4 Macfarlane). P. aeruginosa suspension of 1.2 109 CFU/ml was applied to the inflamed areas of four rat groups and one group was assigned as the negative control group (group 2). Each rat was kept in isolated separate cage to prevent the transmission of infection between animals. At 24 h after inoculation and confirmation of the infection in the injured areas, treatment was started, according to the following group classification: Phosphate-receiving group: Includes 10 infected rats. Phosphate-enriched lotion formulation was applied on the surface of the wounds one time a day. Negative control group: Includes 10 rats that were burnt. P. aeruginosa suspension was not applied, neither did they receive any treatment.
Please cite this article in press as: Mohammadi-Samani S, et al. Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem. Burns (2016), http://dx.doi.org/10.1016/j.burns.2015.09.003
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Positive control group: Includes 10 rats that were burnt. P. aeruginosa suspension was applied, but they did not receive any treatment. Silver-receiving group: Includes 10 rats that were burnt. P. aeruginosa, suspension was applied and they received standard treatment with silver sulfadiazine ointment. Base-receiving group: Includes 10 rats that received only the lotion base with no source of phosphate to evaluate the healing effect of lotion base. Clinical evaluation of the wounds including wound area size and crust formation was performed at the end of each week for 4 weeks.
2.2.5.
Microbiological and histopathological evaluation
On the 28th day, the animals were anesthetized and then decapitated. Sterile culture from wound area was taken to the Microbiology Department immediately and seeded into several plates (thioglycollate, blood agar, chocolate agar, and MacConkey agar) to maximize the number of bacterial species that could be isolated. Organisms were identified using Gram staining, catalase and coagulase, eosin methylene blue (EMB), triple sugar iron agar (TSI), sulfide indole motility (SIM), urea broth, Simmons citrate and methyl red, and Voges– Proskauer broth (MR-VP) test. The wounded skin was carefully excised for histopathological examination. It was fixed in 10% buffered formalin solution and embedded in paraffin blocks. Sections were stained using hematoxylin and eosin method (H&E). Skin tissue sections (5 mm) were prepared and examined under a light microscope (Olympus, Japan). Histopathological evaluation was done by an experienced histopathologist who was blind to treatment conditions. Grading of the findings was done by assigning scores, 0: none; 1: mild; 2: moderate, and 3:
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severe for each of the following criteria: surface ulceration, reepithelialization, granulation tissue, and existence of neutrophil and leukocyte infiltration (sign of acute inflammation) and monocytes/plasma cell (sign of chronic inflammation). Reepithelialization refers to complete epithelialization or wound healing (grade 3), in the absence of ulceration. Any defect in the wound surface indicates that healing was incomplete (grade 1 or 2) and no epithelialization is assigned grade zero [17]. At least five microscopic fields were evaluated to score each specimen.
2.3.
Statistical analysis
Statistical analysis was carried out using SPSS (version 20) Software, USA. All data are expressed as means SD. The obtained data were compared with analysis of variance (ANOVA) technique, followed by Tukey’s comparison tests. P < 0.05 was regarded as statistically significant
3.
Results
Each group consisted of 10 rats. In positive control and silverreceiving groups, three rats died. In base-receiving group, seven animals died during the follow-up period. In microbiological studies, the presence of both gram-positive and gramnegative bacteria such as Klebsiella, Streptococcus, Helicobacter, Citrobacter, and Staphylococcus was confirmed. P. aeruginosa was isolated from all groups except the phosphate-receiving group. Average diameter of the wounds at the beginning of study (day 1) was 1.7 0.2 cm. All of the rats in different groups were controlled during the 28 days of the experiment and the wound healing rate was measured after this period (Fig. 1). In
Fig. 1 – Mean size of the wounds in different groups of the treatment. Please cite this article in press as: Mohammadi-Samani S, et al. Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem. Burns (2016), http://dx.doi.org/10.1016/j.burns.2015.09.003
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Fig. 2 – Histopathological representation of the wounds after 28 days after treatment protocols (H&E 400T); reepithelialization is shown as green arrow. The surface ulceration is marked as yellow arrow. Complete surface ulceration with no re-epithelialization is noticed in burned skin infected with bacteria (positive control), in silver sulfadiazine receiving group, and in lotion base receiving group. Partial re-epithelialization is observed in negative control group in which burned skin was left without any treatment (positive control). Complete surface re-epithelialization with no evidence of surface ulceration was noticed in phosphate enriched lotion receiving group. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)
phosphate-enriched lotion-receiving group, there was significant improvement in wound healing (0.06 0.09 cm), compared to the other groups (P < 0.05). In this group, almost all the ulcers were fully healed, but in groups 3, 4, and 5, there were large open wounds. The wound diameter in the base-receiving group increased (1.9 0.1 cm), but in the positive control and silver-receiving groups that received standard sulfadiazine treatment or were left untreated, no significant increase was observed (P > 0.05) (Fig. 1).
Based on the results given in Fig. 2, microscopic examination of the wound samples revealed that surface ulceration significantly decreased in the phosphate-receiving group, in comparison to that in other groups (P < 0.05). Besides, as shown in Fig. 2, the reepithelialization and granulation tissue formation were significantly better in the phosphate-receiving group, compared to others (P < 0.05). The acute inflammatory cell infiltration decreased in the phosphate-receiving group (P < 0.05), but there was no significant difference in chronic inflammation across the groups (Table 1).
Table 1 – Pathologic score of wound healing in different treatment groups. Surface ulceration G1 G1 G1 G1
vs vs vs vs
G2 G3 G4 G5
15.19 vs 13.58** 9.06 vs 21.75* 9.34 vs 24.75* 11.50 vs 24.10*
Re-epithelialization 13.22 19.84 24.47 25.78
vs vs vs vs
16.21** 7.38* 11.31* 12.68*
Granulation tissue formation 17.25 12.41 15.22 17.94
vs vs vs vs
10.83** 17.29** 18.68** 18.95**
Acute inflammation 16.47 11.53 13.75 14.94
vs vs vs vs
11.88** 18.46* 20.83* 21.35**
Chronic inflammation 16.19 14.59 18.09 21.97
vs vs vs vs
12.25** 14.38** 15.97** 15.73**
Note: All groups were compared with G1. G1: phosphate lotion receiving group; G2: negative control group; G3: positive control group; G4: silver sulfadiazine receiving group; and G5: base lotion receiving group. * Significant (P-value < 0.05). ** Not significant (P-value > 0.05).
Please cite this article in press as: Mohammadi-Samani S, et al. Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem. Burns (2016), http://dx.doi.org/10.1016/j.burns.2015.09.003
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4.
Discussion
The size and Zeta potential of the emulsified phase in phosphate-enriched formulation were less than that of base formulation due to the effect of phosphate ions on modulation of the positive ion intensity in the diffusion layer. In this regard, the net effect of the presence of phosphate ions was a decrease in Zeta potential. In wound healing process, the damaged tissues are restored to their normal condition via several mechanisms including wound contraction that help shrinkage of the wound area and its rate depends on the ability of the tissue to repair. Silver sulfadiazine ointment proved to be an effective antibacterial agent but was shown to delay the healing process in burn wounds due to inhibition of proliferation of the keratinocytes and fibroblast, leading to impairment of the wound healing process [18–20]. During this study, an invasive strain of P. aeruginosa was isolated from the infected burn wound of a patient in Ghotbedin Burn Hospital, Shiraz, southern Iran. An antibiogram test of this strain (PTCC 1811) revealed that it is a multidrug-resistant strain and according to the results, group 4 that received silver sulfadiazine ointment showed no improvement in treatment and P. aeruginosa was not eradicated from the wounds which were more serious than those in positive control group (group 3). On the other hand, group 1, exhibited significant reduction in the wound surface area (P < 0.05) and skin contraction was almost complete. The wounds of the rats in the negative control group were in a better condition than in the positive control group. Phosphate is an essential chemical entity for all living organisms. Phosphate participates in critical biochemical processes that are essential for maintaining energy dynamics in the living cells and is also a principal component of nucleic acids and phospholipids in the cell membrane. Phosphate depletion during major traumatic conditions such as surgery and burn has been reported previously [16]. Exposure of P. aeruginosa to subinhibitory antibiotic concentrations and nutrient-deficient environment leads to remarkable changes in gene expression and alteration in virulence and resistance of this bacterium to antibiotics [21–23]. Gene activation for uptake of inorganic phosphate as well as phosphate acquisition from alternative sources of organic phosphate compensates for phosphate limitation for P. aeruginosa and the consequence of this process is probably change in virulence behavior and swarming motility. Previous studies also confirm huge transcriptional changes in genes related to phosphate acquisition, quorum sensing, chemotaxis, toxin secretion, and regulation when P. aeruginosa is grown in a low-phosphate medium [23]. In this regard, we aimed to supply sufficient quantity of inorganic phosphate to upregulate the expression of different genes involved in phosphate acquisition process. The consequences of this process seemed to change in virulence and swarming behavior of P. aeruginosa. According to the results of this study, phosphate supplementation in an adequate concentration probably triggers transcriptional changes in gene regulation to convert the pathogenic and invasive strain of P. aeruginosa to the noninvasive phenotype. We suggest the use of this strategy as a supportive treatment
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of P. aeruginosa wound infection. In doing so, the need for antibiotic therapy is lessened and would be unnecessary, but further studies are recommended to well establish the usefulness of this new strategy in clinical practices.
Conflict of interest The authors declare that there are no conflicts of interest to disclose.
Acknowledgments The authors acknowledge the financial support of the Shiraz University of Medical Sciences (grant No. 36-4460). The authors wish to thank Hassan Khajehei, PhD, for linguistic editing of the manuscript. The authors also thank Ali Poustforoushfard, Katayoun Javidnia, Mehrdad Vosoughi, Mohammad Taheri, Omid Kouhi, and Hamid Reza Toutounchi for providing technical support.
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Please cite this article in press as: Mohammadi-Samani S, et al. Effects of phosphate supplementation on Pseudomonas aeruginosa invasive behavior in burn wound infections: A simple approach to a big problem. Burns (2016), http://dx.doi.org/10.1016/j.burns.2015.09.003