Evaluation of Mannosidase and Trypsin Enzymes Effects on Biofilm Production of Pseudomonas aeruginosa Isolated from Burn Wound Infections

Biofilm is an important virulence factor in Pseudomonas aeruginosa and has a substantial role in antibiotic resistance and chronic burn wound infections. New therapeutic agents against P. aeruginosa, degrading biofilms in burn wounds and improving the efficacy of current antimicrobial agents, are required. In this study, the effects of α-mannosidase, β-mannosidase and trypsin enzymes on the degradation of P. aeruginosa biofilms and on the reduction of ceftazidime minimum biofilm eliminating concentrations (MBEC) were evaluated. All tested enzymes, destroyed the biofilms and reduced the ceftazidime MBECs. However, only trypsin had no cytotoxic effect on A-431 human epidermoid carcinoma cell lines. In conclusion, since trypsin had better features than mannosidase enzymes, it can be a promising agent in combatting P. aeruginosa burn wound infections.


Introduction
Burn wound infections are one of the most important complications that occur after burn injuries and may be associated with serious clinical complications and increased morbidity and mortality [1,2]. Pseudomonas aeruginosa is one of the most important pathogens involved in burn infections [1]. The emergence of multidrug-resistant P. aeruginosa infections is the major concern with managing P. aeruginosa burn infections as it is very difficult to treat [3]. P. aeruginosa alters the expression of its virulence factors in wound infections [2], including the production of biofilm in burn wounds [4]. Such hospital-acquired infections delayed healing for 2 to 4 weeks [5]. The biofilm mediates bacterial stability and protects them from surrounding environment, such as the immune system and increases the antibiotic resistance [6].
The biofilm matrix in P. aeruginosa is composed of three distinct exopolysaccharides: alginate, Psl and Pel. Alginate is a polymer consisting of β-D-mannuronic acid and α-L-guluronic acid and has a substantial role in structural stability and protection of biofilm. Psl is a polysaccharide composed of a repeating pentasaccharide, consisting of D-mannose, D-glucose and Lrhamnose. Psl is important in the initiation of biofilm formation and protection of biofilm structure. Pel is the third polysaccharide which is present in P. aeruginosa biofilm and is glucose-rich [7]. Additionally, a lot of surface proteins are involved in P. aeruginosa biofilm formation [8].
Due to the increasing P. aeruginosa antibiotic resistance and given the importance of biofilm in increasing the antimicrobial resistance, researchers are exploring novel therapeutic strategies targeting biofilms. This may contribute to improve the treatment of biofilm-related infections [9]. Some of the anti-biofilm methods that have been studied in recent years include: small molecule based inhibitors, phytochemicals, bacteriophage therapy, photodynamic therapy, antimicrobial peptides, monoclonal antibodies, nanoparticles and biofilm degrading enzymes [10][11][12][13].
The α-mannosidase enzyme is an acid hydrolase which is located in plant vacuoles and is thought to be involved with the turnover of N-linked glycoproteins and has been purified from Canavalia ensiformis (Jack bean) [14]. The β-mannosidase enzyme was purified from helix pomatia and hydrolyzes the terminal mannose residues, which are β-1!4 linked to oligosaccharides or glycopeptides [15]. Based on the structure of Psl polysaccharide and due to the performance features of mannosidase enzymes, it was assumed that these enzymes may destroy Psl polysaccharide.
Trypsin is a pancreatic serine endoprotease that cleaves proteins or peptides on the carboxyl side of arginine (R) or lysine (K) residues [16]. It was supposed that trypsin enzyme may destroy protein contents of the biofilm matrix in P.aeruginosa.
In the current study, we investigate the effects of mannosidase and trypsin enzymes on the degradation of biofilms of P. aeruginosa strains that were isolated from burn wound infections.

Bacterial strains
A total number of 57 P. aeruginosa isolates were collected from infections in burn wound patients from Shahid Motahari Hospital of Iran University of Medical Sciences, during October 2013 through March 2014. The identity of the isolates were determined with by conventional biochemical tests including Gram stain, oxidase, catalase, oxidation-fermentation (OF) test and the Kligler Iron Agar (KIA) tests [17].

Ethics Statement
The Central Laboratory from Shahid Motahari Hospital provided the P. aeruginosa isolates for this study. The clinical information presented in this manuscript was obtained from the patient's medical record, considering the sample type. The study protocol was approved by the Ethics Committee of Tehran University of Medical Sciences (No 25137).
The MICs of Amikacin (Sigma Aldrich, St Louis, USA) and Ceftazidime (Jaber Ebne Hayyan Co, Iran) were determined by CLSI broth microdilution method (MIC range,0.5 to 256 μg/ml). P. aeruginosa ATCC 27853 were used as a control for quality assurance of the test.

Detection of genes encoding biofilm exopolysaccharides
The genes encoding biofilm exopolysaccharides (algD, pelF and pslD) were targeted by a PCRbased method, using primers listed in Table 1 [17]. The following protocol was used for PCR procedure. DNA extraction was performed by boiling method. Each 12.5 μL reaction contains: 2.5 μL of DNA, 5 μL of Taq 2× Master Mix (Ampliqon, Denmark), 0.25 μL of each forward and reverse primers with the concentration of 10 pmol/μL and 4.5 μL of distilled water. PCR was performed under the following conditions: initial denaturation for 5 min at 95°C, then denaturation for 1 min at 95°C, 30 cycles of 40 s at 58°C (for algD, pelF and pslB genes), and 56°C (for pslD gene), 45 second at 72°C, and a final elongation step for 5 min at 72°C. PCR products were analyzed with UV light after running at 120V for 45 minute on a 1% agarose gel.

Biofilm assay
P.aeruginosa isolates were inoculated in 5ml trypticase soy broth (TSB) (Gibco, USA) and incubated for 24 h at 37°C, then they were diluted in TSB to a turbidity equal to 0.5 McFarland standard and each well of a flat-bottomed polystyrene 96-well microtiter plate (Tissue culture plate 96 wells, JET BIOFIL, Canada) were inoculated with 100 μL of these dilutions. Pseudomonas aeruginosa ATCC 27853 and sterile broth were used as positive and negative control. After 24 h incubation at 37°C, the supernatant (containing non-adherent cells) was removed and wells were rinsed with normal saline solution (0.90% w/v of NaCl) three times. Biofilms were fixed by 96% ethanol, and then stained with crystal violet (1.5% w/v) for 20 minute, after that unbound stain was removed by washing with tap water. The dye was solubilized in 150 μL of 33% (v/v) acetic acid. The optical densities (OD) of the wells were determined by using a microplate reader (Anthos Labtec instruments, type: 22550) set to 550 nm [17]. All assays were performed in triplicate and repeated three times for each strain.
Three standard deviations above the mean absorbance of negative control were considered as cut-off OD (OD C ). Biofilm formation was categorized by the following formulas: If OD < ODc, the biofilm was not formed (negative), If ODc < OD < 2xODc, the biofilm was weak, If 2xODc < OD < 4xODc, the biofilm was moderate. If 4xODc < OD, the biofilm was strong.
Based on sensitivity to amikacin and ceftazidime, biofilm production and genotypic characteristics, isolates were selected for further studies.

Enzymatic assay
The biofilm detachment assay was performed as described previously [11]. Briefly, after the establishment of biofilms in the wells of a flat-bottomed polystyrene 96-well microtiter plate (Tissue culture plate 96 wells, JET BIOFIL, Canada), biofilms were incubated with different concentrations of enzymes (α-mannosidase and β-mannosidase enzyme concentrations: 0.005, 0.01, 0.015, 0.02 and 0.03 unit/ml and trypsin enzyme concentrations: 0.08, 0.175, 0.35, 0.75 and 1.5 μg/ml), the optimal concentration of each enzyme was selected and used for incubation with biofilms for 1h at 37°C. Well content was removed and washed thrice with sterile saline solution (NaCl, 0.9% w/v) and were stained using the CV assay. The optical densities (OD) of the biofilms were determined at 550 nm by using a microplate reader (Anthos Labtec instruments, type: 22550). Biofilms with no enzyme treatment was used as a positive control and medium without bacteria and enzyme was used as a negative control. The test was performed once with three replications. All enzymes were purchased from Sigma Aldrich (St Louis, USA). Since α-mannosidase and β-mannosidase enzymes used similar buffer conditions, their combined effects on P. aeruginosa biofilm production was analyzed as well. This test was carried out once with three replications.
The bactericidal effect of the enzymes on planktonic cells of P. aeruginosa was evaluated. Firstly, 50 μL of Mueller-Hinton broth (Merck, Germany) was added to each microtiter plate well (Tissue culture plate 96 wells, JET BIOFIL, Canada) and subsequently, 50 μL of bacterial suspension with a final inoculum density of 10 8 CFU/ml was added to each well and was mixed with α-mannosidase, β-mannosidase and Trypsin. The microtiter-plate was incubated for 20h at 37°C, and the effect of enzymes on the bacterial growth was determined with respect to well described turbidity. After evaluating the turbidity in the wells, 20 μL of the suspension with no turbidity was inoculated on the TSA media and incubated,and then presence of colonies was checked, This test was performed 3 times.

Determination of the Minimum Biofilm Eliminating Concentrations (MBECs)
The MBECs of bacterial biofilm cultures for amikacin and ceftazidime were determined according to the method of Amorena et al using the XTT (2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) colorimetric assay with some modifications [19]. Briefly, biofilms were established in the wells of a flat-bottomed polystyrene 96-well microtiter plate (Tissue culture plate 96 wells, JET BIOFIL, Canada). After incubation of bacterial biofilms with 100 μL of serial dilutions of antibiotics at 37°C for 20 h, 50 μL of fresh XTT labeling mixture (Roche, Germany) was added to each well and subsequently incubated for 1 h at 37°C in the dark conditions [20]. The lowest concentration of the antibiotic that inhibited re-growth of the bacteria from the treated biofilm was defined as the MBEC value [21]. This test was conducted 3 times. This experiment was performed on 3 strains 1, 2 and 4, because they were susceptible to ceftazidime and amikacin in planktonic state, but strain 3 that was resistant to amikacin and strain 5 that was resistant to both amikacin and ceftazidime, were not involved in the experiment.

The combined effect of enzymes and ceftazidime on P. aeruginosa biofilms
The combined effect of enzymes and ceftazidime on P. aeruginosa biofilms was determined as described previously [22,23]. Briefly, bacterial biofilms with either 100 μL of ceftazidime or ceftazidime with enzyme; α-mannosidase and β-mannosidase (0.02 unit/ml) or trypsin (0.75 μg/ml) were incubated at 37°C for 20 h. Subsequently, the well content was removed and washed with normal saline. The MBEC values of ceftazidime for biofilm cultures were determined using the XTT reduction assay. This test was performed 3 times.
For morphological and viability studies, cells were seeded at a concentration of 5× 10 4 cells/ well in 100 μL of complete medium into 96-well plates and were incubated for 24h in a humidified atmosphere at 37°C and 6.5% CO 2 .
After 24h, when cells reached 60% confluency, selected concentrations of enzymes were added to the wells. To evaluate the cytotoxic effect of enzymes, morphological changes in cells were assessed by invert microscopy (Olympus 1x70, USA) every hour for the first 4 hours and finally after 24h.
Mitochondrial functions of the cells were evaluated by XTT reduction assay. After 24h exposing to the enzymes, specific amounts of XTT labeling mixture was directly added to the culture wells and after 4h incubation in the dark conditions, the absorbance at 492 nm was measured using a microplate reader (Anthos Labtec instruments, type: 22550).
In the present experiment, the positive control consisted of cells without enzyme exposure and for the negative control, sterile medium was used.

Statistical analysis
Based on normal distribution of variables [i.e. ODs of biofilms before (OD B) and after (ODA) enzyme treatment and their differences (OD B-A)], non-parametric tests such as Wilcoxon Signed Ranks test and Paired-Samples T Test were used for comparison of ODs before and after treatment with enzymes and One-way ANOVA test was used for determination the effects of enzymes on different P.aeruginosa strains. A Kruskal-Wallis test was applied to study the effects of combination of mannosidase enzymes on the biofilms of strains. The differences between ceftazidime MBECs before and after using enzymes were evaluated by Mann-Whitney U test for each strain. A One-way ANOVA test was used for comparing viability (%) of the cells after enzyme assay. A P value <0.05 was considered statistically significant. All tests were performed using online available GraphPad software (http://www.graphpad.com).

Results
The antimicrobial resistance of 57 P.aeruginosa isolates to amikacin (AK), gentamicin (GM), cefepime (CPM), ceftazidime (CAZ), Imipenem (IMP), meropenem (MEM) and polimyxine B (PB) were determined by disk diffusion agar method and results are listed in Table 2. The highest resistance rate was observed against CPM and GM (94.7%) and the lowest resistance rate was seen against PB (0%).
The results of microtiter plate assay demonstrated that 55 strains (96.5%) were biofilm producers in which 30.9% of them produced strong biofilms, 47.3% produced medium biofilms and 21.8% of formed weak biofilms. Only 2 strains (3.5%) were non-producers.
The genotypic and phenotypic characteristics of 5 P. aeruginosa isolates that were selected out of 57 strains were presented in Table 4. Isolates had ability to produce moderate or strong biofilms and all of them were susceptible to polymixine B.
From the results of a single experiment, it was concluded that α-mannosidase, 03B2-mannosidase and trypsin enzymes reduced the ODs of the biofilms (P<0.05) (Fig 1). The most effective concentrations of α -mannosidase, β-mannosidase and trypsin enzymes on biofilms were 0.02 units/ml, 0.02 units/ml and 0.75 μg/ml, respectively (Fig 2). There were no significant differences between the effects of combinations of mannosidase enzymes with effects of each enzyme alone (P>0.05) and in both cases, the enzymes detached the biofilms (Fig 3). The βmannosidase had no bactericidal effect, but α-mannosidase and trypsin were toxic and all tested concentrations killed bacterial cells and no turbidity was seen in the wells and no colonies were present.
The MBEC results for bacterial biofilm are listed in Table 5. All three strains were resistant to ceftazidime in biofilm; however these strains were susceptible to this agent in planktonic state (Table 4). The combination of enzymes and ceftazidime significantly decreased the ceftazidime MBECs (P<0.05) ( Table 6). Biofilm did not affect the susceptibility of strains to amikacin and remained susceptible to this antibiotic.
The morphology of the A-431 human epidermoid carcinoma cell lines after 24h incubation with trypsin enzyme had no changes compared to control cells (Fig 4) and trypsin enzyme had no significant effect on mitochondrial activity and cell viability, however the cytotoxic effect of mannosidase enzymes were high and reduced the mitochondrial function of the cells (Fig 5). Discussion P. aeruginosa is one of the causes of serious infections in burn patients and emergence of multidrug resistant (MDR) isolates of P.aeruginosa in the burn units is an important problem in controlling its infections [25].
In our study, more than 90% of the isolates were resistant to amikacin, gentamicin, cefepime and meropenem and the rate of resistance to ceftazidime and Imipenem were 61% and 83%,  respectively. In overall, 87% of the isolates were MDR. In a study conducted by Anvarinejad et al., [26], resistance level to the amikacin, gentamicin, cefepime and meropenem were similar to our study, however resistance to ceftazidime and imipenem were higher than our study (72% and 98%, respectively). Nikokar et al., reported much lower resistance rate for imipenem, gentamicin and amikacin and 42.3% of their isolates were MDR, which was lower than our study [27]. Probably, discrepancies in the antimicrobial resistance levels in various studies are related to the differences in the patterns of antibiotic consumption in different areas. Therefore, appropriate therapeutic regimen for treatment of P.aeruginosa infections should be selected based on the location of bacterial isolation.
According to the results, 98.4% of the isolates formed biofilm that was similar to the results of Vasiljević et al., [28] and Jabalameli et al., [17], Which reflects the importance of biofilm  [29,30]. It is possible that the differences observed in the prevalence of this gene are because of different prevalent clones in each region. There is no prevalence rate about pslD and pelF genes in other regions, but studies demonstrate that pel gene cluster are conserved among isolates of P.aeruginosa, however psl genes are not present in all isolates [31], which is in agreement with our results.
In recent years, enzymatic debriding agents such as collagenase and Papain-urea are using in burn wound treatment because of their effects on collagen, elastin and fibrin that remove necrotic tissues and accelerate wound healing [32]. Regular debridement also eliminates some parts of the biofilm EPS and force the remaining bacteria to return to the state that they are metabolically active, so the antibiotics and antiseptic compounds would be more effective. In addition, the use of anti-biofilm compounds can help to eliminate biofilms from the wound bed or weaken the matrix and disintegrate the biofilm [5].
As is clear in Fig 2, both mannosidase enzymes were effective and degraded the biofilms of P. aeruginosa strains with various genotypic patterns and altered the state of biofilms from strong to the moderate or negative. Biofilm of strain 5 with genotypic pattern of pelF -, algD + , pslDwas not affected (P>0.05). These results may suggest that mannosidase enzymes do not have any effect on the structure of alginate, since alginate does not have any mannose residues in its structure. The mannosidase enzymes also had same effects on ceftazidime MBECs and reduced significantly. The results of toxicity assay indicated that mannosidase enzymes cause changes in cell morphology and reduce the mitochondrial activity of the cells, and are cytotoxic.
According to our results (Fig 2), the trypsin also destroyed the P. aeruginosa biofilm; however its effect was weaker than mannosidase enzymes. These results may not be due to the poor effect of this enzyme but it may contribute to the fewer protein contents of P. aeruginosa biofilm rather than polysaccharides, since proteins are one of the sub components of biofilm in this bacterium [7]. Trypsin also decreased the ceftazidime MBECs significantly as a result of both enzymatic biofilm degradation and its bactericidal effect. The trypsin did not change the morphology or mitochondrial functions of the cells indicating its non-toxicity.
In conclusion, our finding (Fig 2) demonstrated that trypsin enzyme can be a good candidate for future studies in the field of antibiofilm agents, because it could destroy biofilms of P. aeruginosa burn isolates and result in decreased ceftazidime MBECs with no toxic side effects.