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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 4  |  Page : 146-152

Comparison of inhibitory effect between DL–tryptophan and lactoferrin on Pseudomonas aeruginosa biofilm formation in wound dressing


Faculty of Medicine, Tanta University, Egypt

Date of Submission23-May-2019
Date of Decision17-Jun-2019
Date of Acceptance20-Jul-2019
Date of Web Publication02-Aug-2019

Correspondence Address:
Maii Atef Shams Eldeen
Faculty of Medicine, Tanta University
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2221-6189.263707

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  Abstract 


Objective: To compare the inhibitory effect between DL-tryptophan and bovine lactoferrin on biofilm formed by isolated Pseudomonas aeruginosa strains.
Methods: The study was carried out on 40 patients suffering from surgical site infection. Wound pus was collected using sterile swabs after isolation, and identified by common bacteriological methods. Isolated Pseudomonas aeruginosa strains were grown on biofilm enhancing materials, and then the inhibitory effects of different concentrations of DL-tryptophan and lactoferrin were tested using scanning electron microscopy and microtitre plate methods.
Results: There was no significant difference in the inhibitory effect between DL-tryptophan and lactoferrin at 0.5 mg/mL. While in concentration of 1 mg/mL and 2 mg/mL, tryptophan showed more significant inhibitory effect than lactoferrin.
Conclusions: Both DL-tryptophan and bovine lactoferrin have inhibitory effect on Pseudomonas aeruginosa biofilm formation in a dose dependent manner, and the inhibitory effect of DL- tryptophan is stronger.

Keywords: Pseudomonas aeruginosa, Biofilm, Tryptophan, Lactoferrin


How to cite this article:
Elfeky EH, Eldeen MA, Hashish AA, Hassan AM. Comparison of inhibitory effect between DL–tryptophan and lactoferrin on Pseudomonas aeruginosa biofilm formation in wound dressing. J Acute Dis 2019;8:146-52

How to cite this URL:
Elfeky EH, Eldeen MA, Hashish AA, Hassan AM. Comparison of inhibitory effect between DL–tryptophan and lactoferrin on Pseudomonas aeruginosa biofilm formation in wound dressing. J Acute Dis [serial online] 2019 [cited 2019 Sep 22];8:146-52. Available from: http://www.jadweb.org/text.asp?2019/8/4/146/263707




  1. Introduction Top


Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen, and is usually linked to nosocomial infections like burns and surgical site infections[1]. The ability of P. aeruginosa to form biofilm is a key factor for organism to cause persistent infections. The biofilm matrix also leads to significant antimicrobial resistance[2].

The biofilm is composed of microorganisms that grow in an extracellular matrix of proteins, DNA and polysaccharides resulting from microbial metabolism[3]. The biofilm formation occurs in three stages: adhesion, aggregation and maturation. The adhesion stage occurs within the first 4 h of growth, through which microorganisms attach to the inert surfaces by electrostatic force. Then aggregation stage starts 6-12 h post-inoculation. Lastly, after 12 h, the mature biofilm is formed[4]. This biofilm matrix acts as a shield that protects organisms against any protozoan grazers, toxins or immunity attacks. Moreover, it allows diffusion of oxygen, nutrients and wastes to get through[5].

Various studies using scanning electron microscopy (SEM), documented that the existence of bacterial biofilm on wound dressings, medical implants and surgical sutures[6],[7],[8] can develop more antibiotic resistance, leading to additional wound debridement, and further the delayed healing process[9]. Many antibiotics have significantly decreased efficacy against biofilm as compared to planktonic (i.e., free-floating) cells. The extracellular matrix provides a mechanical shield, preventing most antibiotics from reaching their target. Furthermore, biofilm tolerance is depended on the physiological status of biofilm cells, which is characterized by low activity of cell processes such as cell wall, protein, or DNA biosynthesis. Thus, many antibiotics that target those processes are barely active against cells in biofilms[10].

The dramatic increase in the number of multi drug resistant bacteria such as P. aeruginosa, together with the failure of some of the most powerful antibiotics to treat life-threatening infections have created a global health crisis with insistent clinical need for innovative topical approaches to prevent biofilm formation and induce its disassembly in chronic wounds[11].

Biofilm inhibitors include antimicrobial peptides, metal chelators, quorum sensing inhibitors, and amino acids[12]. The aim of this study is to investigate and compare the inhibitory effects between the two natural substances on the biofilm formed by isolated P. aeruginosa strains from wound infections. The first substance is tryptophan (DL-trp) which is an amino acid with beneficial effect on wound healing[13]. The second substance is bovine lactoferrin (bLF) which is a glycoprotein secreted in some body fluids such as tears, semen, vaginal secretions, and milk[14]. LF has shown to have antimicrobial activity against different pathogens[15].


  2. Materials and methods Top


The present study was carried out on patients admitted to Surgical and Burn Units of Tanta University Hospitals. It was conducted in Medical Microbiology and Immunology Department, Faculty of Medicine, Tanta University from March to August 2017.

Ethical approval for this study was provided by the Ethics and Research Committee, Tanta Faculty of Medicine. Written informed consent was obtained from all participants in this research. A code number was put to each sample for adequate provision to maintain privacy of participants and confidentiality of data. The protocol number was 31247/12/16 and the date of approval by the ethics committee was December 2016.

2.1. Subjects

This study included 40 clinically suspected patients who had evidence of local signs for wound infection (Redness, hotness, swelling, purulent discharge or delayed healing of wound) and/or systemic manifestations (fever, chills, or hypotension) with no other apparent source of infection except the wound. Patients who received antibiotics 7 d preceding the study were excluded.

2.2. Specimen collection

All samples were collected with sterile swabs in sterile containers under complete aseptic conditions. Swabs were introduced into the depth of lesion and rolled to aspirate pus or exudation from the wound. Samples were transported as soon as possible to Medical Microbiology and Immunology Department, Faculty of Medicine, Tanta University [Figure 1].
Figure 1: The study flow chart.

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2.3. Specimen processing

Swabs were cultured on nutrient agar and MacConkey agar (Oxoid, UK) for evaluation of the colony size, shape, edge, color, opacity, elevation and surface. The organisms showing characteristic colony morphology of P. aeruginosa were obtained followed by microscopic identification by Gram stained films to clarify the morphology of the bacterial cells (size, shape and arrangement). P. aeruginosa was identified as Gram-negative bacilli of variable size, non-sporing and non-capsulated. Biochemical identification was performed with a simplified scheme of biochemical tests such as sugar fermentation test, triple sugar iron test and oxidase test[16].

2.4. Material preparation

According to the manufacture instructions, M63 Medium (VWR, Germany) & Bacto tryptic soy broth (TSB) (Sigma Aldrich) were used for overnight bacterial growth and biofilm experiments on isolated P. aeruginosa strains. All the isolated seventeen P. aeruginosa strains were inoculated in TSB, with concentration of ½ McFarland (using automatic Turbidemeter) and incubated aerobically for 24 h at 37 °C under static conditions. These inocula were then diluted with M63 minimal medium in a ratio of 1:10[17].

DL-Trp (Alfa Aesar, Germany) and lactoferrin from bLF (Sigma Aldrich, Germany) were prepared to be used as biofilm inhibitory substances. DL-Trp was dissolved in sterile water at 85 °C. Three concentrations of 0.5 mg/mL, 1 mg/mL and 2 mg/mL were prepared. Similarly, bLF was dissolved in sterile water at room temperature. Three concentrations of 0.5 mg/mL, 1 mg/mL and 2 mg/mL were prepared as well. In a microplate, wound dressings were cut into 8 mm rounded discs and soaked in the P. aeruginosa inoculum. Firstly, the negative control well was filled with 0.2 mL TSB. Wells were done for each strain as follow: In the first well, 200 μL of the inoculum and 100 μL sterile broth were added to the dressing and was considered a positive control. In the next 3 wells, 200 μL of the inoculum and 100 μL of bLF at 0.5, 1 and 2 mg/mL were added. Another 200 μL of the inoculum and 100 μL of DL-Trp at 0.5, 1 and 2 mg/mL were added to the dressings in the last 3 wells.

2.5. Detection of inhibitory effect of DL-Trp and bLF on biofilm formation

2.5.1. SEM method

The wound dressings were prepared for SEM investigation. The plate was covered and incubated aerobically at 37 °C for 24 h, then the dressings were taken and washed gently by a micropipette with PBS for 3 times and inserted separately in 2.5% buffered glutaraldehyde + 2% paraformaldehyde in 0.1 M sodium phosphate buffer at pH 7.4 for 2 h for fixation[18]. After fixation, each dressing was put in 1% osmic acid for 90 min then washed three times with PBS (10 min each), then dehydrated with ascending series of ethyl alcohol (30%, 50%, 70%, 90% and absolute alcohol) infiltrated with acetone, each concentration for 30 min. For SEM, dressing was dried with sample preparation equipment (SPI supplies), critical point drying machine using liquid Co. Then the dressing was mounted on aluminum stub and coated with gold-palladium membranes in SPI-MODULE Carbon coater Photos were obtained using a Jeol JSM- 6510 L.V SEM. The microscope was operated at 30 KV at EM Unit, Mansoura University, Egypt. Images were acquired mainly at a magnification of2 000x with some other magnifications (5 000x, 7 000x and 10 000x).

2.5.2. Microtitre plate method

Isolates from fresh agar plates were inoculated in 10 mL TSB and incubated for 24 h at 37 °C, diluted (1:100) with fresh medium. The wells of 96 micro-plates were filled with 0.2 mL of different solutions. At first the negative control well was filled with 0.2 mL TSB. The positive control well was added with 0.2 mL of diluted inoculum of each isolated strain. Then other 6 wells were filled with 0.1 mL of the diluted inocula and 0.1 mL of the tested materials at 0.5, 1 and 2 mg/mL (DL-Trp or bLF). The tissue culture plates were incubated for 24 h at 37 °C . Then the well contents were gently removed by plate tapping and washed four times with 0.2 mL of PBS to remove free-floating planktonic bacteria; wells were dried for 1 h at 60 °C then 0.2 mL of 1% solution of crystal violet was added to each well (this dye stains the cells but not the polystyrene) plates. The plates were incubated at room temperature for 15 min, rinsed thoroughly and repeatedly 3-4 times for about one minute with water, and then the biofilm was quantified after addition of 0.2 mL of 95% ethanol. The optical density (OD) of the wells was measured at 570 nm using auto reader. The percentage inhibition of biofilm activity was calculated using the following equation[19]: Biofilm inhibition (%) = 1- (absorbance of cells treated with DL-Trp or bLF / absorbance of non-treated wells) x 100%. Based on the result of microtitre plate method, a proposed grading was used to detect the degree of biofilm inhibition: 0%-35% is considered a weak effect, 35%-70% represents a moderate effect and more than 70% represents a strong effect.

2.6. Statistical analysis

The data of this study were collected, tabulated and statistically analyzed using SPSS 20. Chi-square test was used to compare between the two substances used.


  3. Results Top


3.1. Infection rate and distribution of isolated bacteria

By culturing on nutrient and MacConkey agar plates, 92.5% of patients were culturally positive and 7.5% of patients were clinically suspected but without microbial growth on ordinary media.

Among the 40 patients, 11 had mixed infections including 8 cases infected with two microorganisms and 3 cases infected with three microorganisms. A total of 51 strains were isolated. P. aeruginosa was the predominant microorganism (33.3%) followed by Klebsiella (27.5%), Escherichia coli (15.7%), Candida (5.9%), Acinetobacter (3.9%) and Proteus (3.9%).

3.2. Inhibitory effect of DL-Trp and bLF on P. aeruginosa biofilm formation

All examined strains were strong biofilm producers. When DL- Trp and bLF were used in a concentration of 0.5 mg/mL, the biofilm formation was moderately restricted in most of examined strains treated by DL-Trp while it was weakly inhibited by bLF for most of the examined strains as shown in [Table 1], [Figure 2]B and [Figure 3]B compared to positive control (P. aeruginosa strain without inhibitory substance).
Table 1: Inhibitory effect of DL-tryptophan and lactoferrin at different concentration [n(%)].

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Figure 2: SEM result of P. aeruginosa strain treated with DL-tryptophan (x2 000). A: positive control (strong biofilm producer without being treated with any inhibitory substance); B: treated with DL-tryptophan at 0.5 mg/mL; C: treated with DL-tryptophan at 1 mg/mL; D: treated with DL-tryptophan at 2 mg/mL. The biofilm formation was moderately restricted at 0.5 mg/mL, strongly restricted at 1 mg/mL. mostly completely restricted at 2 mg/mL. P. aeruginosa is indicated by the yellow arrows, while the bacterial biofilm is indicated by the red ones.

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Figure 3: SEM result of P. aeruginosa strain treated with lactoferrin (x2 000). A: positive control (strong biofilm producer without being treated with any inhibitory substance); B: treated with lactoferrin at 0.5 mg/mL; C: treated with lactoferrin at 1 mg/mL; D: treated with lactoferrin at 2 mg/mL. The biofilm formation was weakly inhibited at 0.5 mg/mL, moderately restricted at 1 mg/mL, strongly (but not completely) restricted at 2 mg/mL. P. aeruginosa is indicated by the yellow arrows, while the bacterial biofilm is indicated by the red ones.

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At 1 mg/mL, the biofilm formation was strongly restricted in most cases treated with DL-Trp while it was moderately restricted by bLF in most of the examined strains as shown in [Table 1], [Figure 2]C and [Figure 3]C.

Both DL-Trp and bLF at 2 mg/mL had a strong inhibitory effect on biofilm formation. DL-Trp significantly inhibited the biofilm formation in most cases and moderate in other cases while it was slightly less inhibited by bLF as shown in [Table 1], [Figure 2]D and [Figure 3]D.

Our results showed that the inhibitory effect was similar between DL-Trp and bLF at 0.5 mg/mL, while at 1 mg/mL and 2 mg/mL, DL-Trp showed stronger inhibitory effect than bLF [Figure 4].
Figure 4: Inhibitory effect of both DL-tryptophan and lactoferrin on biofilm formation according to microtitre plate method.

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  4. Discussion Top


Wound infection is a serious problem in all surgical fields. Within 24 h, patients would suffer from opportunistic bacterial attacks that vary from simple infections. Some are caused by complicated bacteria, which are multi drug resistant or biofilm forming bacteria such as P. aeruginosa.

Our study investigated 40 wound swabs, 51 strains were isolated, 43 (84.3%) of them were Gram negative bacteria. In agreement with our result, an Egyptian study[20] reported the predominance of Gram-negative isolates (78%) in a surveillance study at Alexandria University Hospital. Other studies[21],[22] also reported that the primary pathogenic group isolated from infected wounds in their study patients was Gram-negative bacteria. In contrast to our results, a study reported that aerobic Gram-positive bacteria represented 57.6% of the isolates, while aerobic Gram-negative bacteria represented 42.4% of the isolates[23]. Many factors can contribute to the high rate of infection with Gram-negative bacteria. The hands of healthcare personnel may act as the primary source for transmission of Gram-negative bacteria, especially when the skin is damaged or kept moist together with poor hand hygiene compliance.

In this study, P. aeruginosa was predominant among Gram negative isolates (33.3%). This can be explained by its ability to produce many virulence factors that mediate several pathogenic mechanisms, including adhesion, nutrient acquisition, immune system evasion, leukocyte killing, tissue destruction, and bloodstream invasion. In agreement with our study, it was reported that the most common isolated organism was P. aeruginosa (19.4%)[24]. On the other hand, another screening study[25] revealed that Staphylococcus was the predominant organism identified in 66.7% of patients. Also a Cameroonian study[26] reported that Staphylococcus aureus was the most common isolate (24.8%) followed by P. aeruginosa (23.1%). This can be explained by the different sampling source.

In the present study, all P. aeruginosa isolates (100%) were biofilm producers. In agreement with our study, previous studies[27],[28],[29] reported that all examined isolates were biofilm producers. On the other hand, an Egyptian study[30] reported that only 22.2% of isolates were biofilm producers, while 77.8% of isolates didn’t form biofilm. Inability of some P. aeruginosa strains to form biofilm may be attributed to genetic mutations in the lasR and rhlR quorum sensing genes that lead to decreased virulence and inhibited biofilm production as detected by sequencing results in a study by Lima et al.[31].

Our selection of DL-Trp was based on the results reported by previous study[17] which stated that D and L isoforms of tryptophan are equally effective in inhibiting P. aeruginosa biofilm formation. In our study, it was detected that DL-Trp had an inhibitory effect on P. aeruginosa biofilm in in-vitro wound dressing in a dose-dependent manner. Similarly, Brandenburg et al.[17] revealed that DL-Trp inhibited P. aeruginosa biofilm formation on the wound dressing in a dose dependent manner from 0.5 to 10 pM. Also, the inhibitory effect of tryptophan at 0.05 μM-50 μM on P. aeruginosa biofilm formation was observed by Gnanadhas et al.[32].

In this study DL-Trp at 0.5 mg/mL moderately inhibited the biofilm formed by P. aeruginosa, moderately to strongly restricted at 1 mg/mL and strongly inhibited at 2 mg/mL. In comparison with our study, it was reported that combinations of DL-Trp inhibited biofilm formation at 24 h, 82% at 0.6 mg/mL, 71% at 1 mg/mL and 93% at 2 mg/mL[17]. The study also reported the significant inhibition by DL-Trp above 5 mM (1 mg/mL). These differences may be related to the different spp of P. aeruginosa and also manual skills. In partial agreement, another study[33] approved that DL-Trp showed an inhibitory effect on biofilm production at 3 mM (0.6 mg/mL) with approximately 65% and in concentration of 10 mM (2 mg/mL) the inhibition represented about 82%. In this study SEM examination showed that tryptophan at high concentration inhibited the bacterial growth. In agreement with our result, it was[11] approved that DL-Trp significantly decreased bacterial colonization of P. aeruginosa on the dressing at concentrations above 5 mM (>1 mg/mL). Rumbo et al.[34] who tested the effect of different D-amino acids on bacterial growth and biofilm formation, reported that in the presence of D-Trp at 40 mM, the bacterial growth of P. aeruginosa was delayed by about 25%.

In the current study, an inhibitory effect of bLF on biofilm formation was detected in all the tested clinical isolates, but with different degrees and in a dose-dependent manner. However, bacterial growth inhibition of bLF at all concentrations was not observed. Higher bLF concentration (>2 mg/mL) may be used to inhibit the growth of planktonic cells of P. aeruginosa, as reported previously[35]. In agreement with this result, it was reported that LF inhibited biofilm by P. aeruginosa formation in a dose-dependent manner (0.1 mg/mL-2.0 mg/mL), but concentrations exceeding 4 mg/mL showed less inhibition[36]. These results indicate that an optimal concentration of LF may exist with respect to the inhibition of biofilm formation. In agreement with our result, Singh[35] reported that LF at 20 μg/mL did not affect the growth rate of P. aeruginosa in the medium. At sub-inhibitory concentrations of LF (20 μg/mL), the bacteria attached and multiplied, but they failed to form micro-colonies or differentiated biofilm structures. Lactoferrins’ bacteriostatic function was explained by its ability to take up the Fe3+ ion, limiting use of this nutrient by bacteria at the infection site and inhibiting the growth of these microorganisms as well as the expression of their virulence factors[37].

In a partial agreement with the current study, Xu et al.[38] approved that bLF in serial dilutions (100 to 0.39 μmol/L) had a bactericidal activity against P. aeruginosa and also decreased biofilm formation, both growing and static in a dose-dependent manner. Chen et al.[39] also reported that immobilized LF was able to reduce the adhesion of the strains of these species and there for inhibit biofilm formation. A Japanese study[40] on Porphyromonas gingivalis and Prevotella intermedia indicated that bLF at 0.008 mg/mL to 2 mg/mL had the ability to suppress the growth of planktonic Porphyromonas gingivalis and Prevotella intermedia independently of the iron-bound form of bLF. Furthermore, they found that various iron-bound forms of bLF can inhibit the biofilm formation of these bacteria even at lower concentrations and that LF alone or in combination with antibiotics can reduce the amounts of preformed biofilms of these bacteria.

The current study compared the effect between DL-Trp and bLF on biofilm formed by P. aeruginosa on wound dressing, and showed difference in the effect. Moreover, it is was reported that the inhibitory effect could be detected until optimum concentration[37].

There were some limitations and shortcomings. Firstly, the study could have been generalized, if further samples were collected from other departments beside the surgical department as further sources of P. aeruginosa infection. Moreover, it would be more conclusive, if we investigated larger number of patient samples for better statistical analysis. In this study, qualitative method of SEM was intensively utilized and delivered satisfactory results identifying the biofilm structure and inhibitory effect. Moreover, quantitative methods such as microtitre plate method can provide better evaluation.

Conflict of interest statement

The authors report no conflict of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
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  2. Materials and...
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Abstract
1. Introduction
3. Results
4. Discussion
References
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