|Year : 2019 | Volume
| Issue : 4 | Page : 146-152
Comparison of inhibitory effect between DL–tryptophan and lactoferrin on Pseudomonas aeruginosa biofilm formation in wound dressing
Eman Hussein Hussein Elfeky, Maii Atef Shams Eldeen, Amel Abd El-Tawab Hashish, Azza Mahmoud Hassan
Faculty of Medicine, Tanta University, Egypt
|Date of Submission||23-May-2019|
|Date of Decision||17-Jun-2019|
|Date of Acceptance||20-Jul-2019|
|Date of Web Publication||02-Aug-2019|
Maii Atef Shams Eldeen
Faculty of Medicine, Tanta University
Source of Support: None, Conflict of Interest: None
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|| |
Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen, and is usually linked to nosocomial infections like burns and surgical site infections. 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.
The biofilm is composed of microorganisms that grow in an extracellular matrix of proteins, DNA and polysaccharides resulting from microbial metabolism. 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. 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.
Various studies using scanning electron microscopy (SEM), documented that the existence of bacterial biofilm on wound dressings, medical implants and surgical sutures,, can develop more antibiotic resistance, leading to additional wound debridement, and further the delayed healing process. 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.
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.
Biofilm inhibitors include antimicrobial peptides, metal chelators, quorum sensing inhibitors, and amino acids. 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. The second substance is bovine lactoferrin (bLF) which is a glycoprotein secreted in some body fluids such as tears, semen, vaginal secretions, and milk. LF has shown to have antimicrobial activity against different pathogens.
| 2. Materials and methods|| |
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.
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].
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.
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.
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. 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: 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|| |
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(%)].|
Click here to view
|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.|
Click here to view
|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.|
Click here to view
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.|
Click here to view
| 4. Discussion|| |
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 reported the predominance of Gram-negative isolates (78%) in a surveillance study at Alexandria University Hospital. Other studies, 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. 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%). On the other hand, another screening study revealed that Staphylococcus was the predominant organism identified in 66.7% of patients. Also a Cameroonian study 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,, reported that all examined isolates were biofilm producers. On the other hand, an Egyptian study 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..
Our selection of DL-Trp was based on the results reported by previous study 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. 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..
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. 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 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 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. 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. 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. 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 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.
In a partial agreement with the current study, Xu et al. 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. 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 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.
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.
| References|| |
Bangera D, Shenoy SM, Saldanha DR. Clinico-microbiological study of Pseudomonas aeruginosa
in wound infections and the detection of metallo- β -lactamase production. Int Wound J
2016; 13(6): 1299-1302.
Billings N, Millan MR, Caldara M, Rusconi R, Tarasova Y, Stocker R, et al. The extracellular matrix component Psl provides fast-acting antibiotic defense in Pseudomonas aeruginosa
biofilms. Plos Pathog
2013; 9(8): e1003526.
Percival SL, McCarty SM, Lipsky B. Bioflms and wounds: an overview of the evidence. Adv Wound Care
2015; 4(7): 373-381.
Arnold WV, Shirtliff ME, Stoodley P. Bacterial biofilms and periprosthetic infections. J Bone Joint Surg Am
2013; 95(24): 2223-2229.
Song F, Koo H, Ren D. Effects of material properties on bacterial adhesion and biofilm formation. J Dent Re
2015; 94(8): 1027-1034.
Henry-Stanley MJ, Hess DJ, Barnes AM, Dunny GM, Wells CL. Bacterial contamination of surgical suture resembles a biofilm. Surg infect
2010; 11(5): 433-439.
Lipp C, Kirker K, Agostinho A, James G, Stewart P. Testing wound dressings using an in vitro
wound model. J Wound Care
2010; 19(6): 220-226.
Edmiston CE Jr, Krepel CJ, Marks RM, Rossi PJ, Sanger J, Goldblatt M, et al. Microbiology of explanted suture segments from infected and noninfected surgical patients. J Clin Microbiol
2013; 51(2): 417-421.
Roche ED, Renick PJ, Tetens SP, Ramsay SJ, Daniels EQ, Carson DL. Increasing the presence of biofilm and healing delay in a porcine model of MRSA-infected wounds. Wound Repair Regen
2012; 20(4): 537-543.
Otto M. Biofilms in Disease. In: Rumbaugh KP, Ahmad I (Eds.). Antibiofilm agents. From diagnosis to treatment and prevention
. Heidelberg: Springer; 2014, p. 3-13.
Brandenburg KS, Calderon DF, Kierski PR, Brown AL, Shah NM, Abbott NL, et al. Inhibition of Pseudomonas aeruginosa
biofilm formation on wound dressings. Wound Repair Regen
2015; 23(6): 842-854.
Sanchez CJ, Akers KS, Romano DR, Woodbury RL, Hardy SK, Murray CK, et al. D-amino acids enhance the activity of antimicrobials against biofilms of clinical wound isolates of Staphylococcus aureus
and Pseudomonas aeruginosa. Antimicrob Agents Chemother
2014; 58(8): 4353-4361.
Ito H, Ando T, Ogiso H, Arioka Y, Saito K, Seishima M. Inhibition of indoleamine 2, 3-dioxygenase activity accelerates skin wound healing. Biomaterials
2015; 53: 221-228.
Park JH, Park GT, Cho IH, Sim SM, Yang JM, Lee DY. An antimicrobial protein, lactoferrin exists in the sweat: proteomic analysis of sweat. Exp Dermatol
2011; 20(4): 369-371.
Gifford JL, Ishida H, Vogel HJ. Structural characterization of the interaction of human lactoferrin with calmodulin. PLoS One
2012; 7(12): e51026.
Cheesbrough M. Tropical countries
. Part 2. 2nd ed. London: Cambridge University Press. 2007; 7: 234-237.
Brandenburg KS, Rodriguez KJ, McAnulty JF, Murphy CJ, Abbott NL, Schurr MJ, et al. Tryptophan inhibits biofilm formation by Pseudomonas aeruginosa. Antimicrob Agents Chemother
2013; 57(4): 1921-1925.
Ansari MA, Khan HM, Khan AA, Cameotra SS, Alzohairy MA. Anti-biofilm efficacy of silver nanoparticles against MRSA and MRSE isolated from wounds in a tertiary care hospital. Indian J Med Microbiol
2015; 33(1): 101-109.
Abdel Rahim KA, Ali Mohamed AM. Bactericidal and antibiotic synergistic effect of nanosilver against Methicillin-Resistant Staphylococcus aureus. Jundishapur J Microbiol
2015; 8(11): e25867.
Hafez S, Saied T, Hasan E, Elnawasany M, Ahmad E, Lloyd L, et al. Incidence and modifiable risk factors of surveillance of surgical site infections in Egypt: A prospective study. Am J Infec Cont
2012; 40(5): 426-430.
Sohn AH, Tien NP, Mai VT, Van Nho V, Hanh TN, Ewald B, et al. Microbiology of surgical site infections and associated antimicrobial use among Vietnamese orthopedic and neurosurgical patients. Infec Control Hosp Epidemiol
2006; 27(8): 855-862.
Leong WJ, Hasan H, Zakaria Z, Ghazali MZ, Ab Hamid SA, Hassan SA. Risk factors and etiologies of clean and clean contaminated surgical site infections at a tertiary care center in Malaysia. South Asian J Trop Med Public Health
2017; 48(6): 1299-1307.
Wong SY, Manikam R, Muniandy S. Prevalence and antibiotic susceptibility of bacteria from acute and chronic wounds in Malaysian subjects. J Infec Dev Ctries
2015; 9(9): 936-944.
Hefni AA-H, Ibrahim A-MR, Attia KM, Moawad MM, El-ramah AF, Shahin MM, et al. Bacteriological study of diabetic foot infection in Egypt. J Arab Soci Med Resear
2013; 8: 26-32.
Sporer SM, Rogers T, Abella L. Methicillin-Resistant and Methicillin- Sensitive Staphylococcus aureus
screening and decolonization to reduce surgical site infection in elective total joint arthroplasty. J Arthroplasty
2016; 31(9 Suppl): 144-147.
Kihla AJ-FT, Ngunde PJ, Evelyn MS, Gerard N, Ndip RN. Risk factors for wound infection in health care facilities in Buea, Cameroon: aerobic bacterial pathogens and antibiogram of isolates. Pan Afr Med J
2014; 18: 6.
Abbas HA, Serry FM, EL-Masry EM. Combating Pseudomonas aeruginosa
biofilms by potential biofilm inhibitors. Asian J Res Pharm Sci
2012; 2: 66-72.
Abd El Galil K, AbdelGhani S, Sebak MA, El-Naggar W. Detection of biofilm genes among clinical isolates of Pseudomonas aeruginosa
recovered from some Egyptian hospitals. N Egypt J Microbiol
2013; 36: 86-101.
Pastar I, Nusbaum AG, Gil J, Patel SB, Chen J, Valdes J, et al. Interactions of methicillin resistant Staphylococcus aureus
USA300 and Pseudomonas aeruginosa
in polymicrobial wound infection. PLoS One
2013; 8(2): e56846.
Mohamad EA, El Shalakany AH. Detection of biofilm formation in uropathogenic bacteria. Egypt J Med Microbiol
2015; 24: 49-58.
Lima JLdC, Alves LR, Jacomé PRLdA, Bezerra Neto JP, Maciel MAV, Morais MMCd. Biofilm production by clinical isolates of Pseudomonas aeruginosa
and structural changes in LasR protein of isolates non biofilm-producing. Braz J Infect Dis
2018; 22(2): 129-136.
Gnanadhas DP, Elango M, Datey A, Chakravortty D. Chronic lung infection by Pseudomonas aeruginosa
biofilm is cured by L-Methionine in combination with antibiotic therapy. Sci Rep
2015; 5: 16043.
Li XH, Kim SK, Lee JH. Anti-biofilm effects of anthranilate on a broad range of bacteria. Sci Rep
2017; 7(1): 8604.
Rumbo C, Vallejo JA, Cabral MP, Martínez-Guitián M, P
érez A, Beceiro A, et al. Assessment of antivirulence activity of several d-amino acids against Acinetobacter baumannii
and Pseudomonas aeruginosa. J Antimicrob Chemother
2016; 71(12): 3473-3481.
Singh PK. Iron sequestration by human lactoferrin stimulates P. aeruginosa
surface motility and blocks biofilm formation. Biometals
2004; 17(3): 267-270.
Kamiya H, Ehara T, Matsumoto T. Inhibitory effects of lactoferrin on biofilm formation in clinical isolates of Pseudomonas aeruginosa. J Infect Chemother
2012; 18(1): 47-52.
Redwan EM, El-Baky NA, Al-Hejin AM, Baeshen MN, Almehdar HA, Elsaway A, et al. Significant antibacterial activity and synergistic effects of camel lactoferrin with antibiotics against methicillin-resistant Staphylococcus aureus
(MRSA). Res Microbiol
2016; 167(6); 480-491.
Xu G, Xiong W, Hu Q, Zuo P, Shao B, Lan F, et al. Lactoferrin-derived peptides and lactoferricin chimera inhibit virulence factor production and biofilm formation in Pseudomonas aeruginosa. J Appl Microbiol
2010; 109(4): 1311-1318.
Chen R, Cole N, Dutta D, Kumar N, Willcox MDP. Antimicrobial activity of immobilized lactoferrin and lactoferricin. J Biomed Mater Res B Appl Biomater
2017; 105(8): 2612-2617.
Wakabayashi H, Yamauchi K, Kobayashi T, Yaeshima T, Iwatsuki K, Yoshie H. Inhibitory effects of lactoferrin on growth and biofilm formation of Porphyromonas gingivalis
and Prevotella intermedia. Antimicrob Agents Chemother
2009; 53(8): 3308-3316.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]