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International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: 0974-4304, ISSN(Online): 2455-9563 Vol.9, No.12, pp 424-431, 2016
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Antibiofilm Effect of Biopolymer Dextran – Gentamycin- PVP Blend in Catheters
Jehan Abdul Sattar Salman* and Mustafa Z. Salim
Department of Biology, College of Science, Al-MustansiriyahUniversity, Baghdad-Iraq.
Abstract : Objectives: The objective of this study was to determine the antibiofilm effect of biopolymer dextran produced by Leuconostoc mesenteroides ssp. Mesenteroides and their blends with gentamycin and Polyvinylpyrrolidone(PVP)against pathogenic bacteria in catheters.
Methods: Minimum inhibitory concentration (MIC) values of dextran, gentamycin and Polyvinylpyrrolidone(PVP) were determined against bacteria isolated from catheters. Antibiofilm effect of biopolymer dextran and its blenders (gentamycin – PVP) was determined alone and as blends (dextran- gentamycin), (dextran-PVP), (dextran - gentamycin -PVP) using a pre-coated method in micro titer plate and catheters.
Results:The MIC of dextran was found to be 32mg/ml for E.coli , P.aeruginosa, S.aureus isolates , the MIC for P.mirabilis isolates was between (16 –32 ) mg/ml while in S.epidermidis was between (32 – 64)mg/ml. The MIC of gentamycin, PVP was found to be 16 µg/ml, 256 mg/ml respectively for all bacterial isolates. Biopolymer blend shad the ability to inhibit biofilm formation in micro titer plate and catheters, the highest biofilm inhibition ratio 80% was recorded of biopolymer dextran - gentamycin –PVP blend against S.epidermidis(Se1) in microtiterplate, while in catheters the same biopolymer blend had antibiofilm effect with biofilm inhibition ratio reached to 90% and 81% against E.coli(E2) and S. aureus(Sa2) respectively after 72h.
Conclusion: The biopolymer dextran- gentamycin – PVP blend had antibiofilm properties against pathogenic bacteria isolated from catheters. Also had a potential to be used as antibiofilm coating for catheters.
Keywords : Dextran,Polyvinylpyyrodine, Gentamycin, Antibiofilm, Catheter.
Introduction
The most common medical device infection is the catheter- acquired urinarytract infection where the period of time of catheters insertion to the body is quite enough for bacterial growth in the catheter1,2. Catheters provide a suitable surface for bacterial attachment where 24 hours is enough for bacterial attachment and can easily enter urinary system3. The most important cause of bacteriuria along the catheter surface is the ability of these pathogens to form the biofilm where bacteria can attach quickly to the surface of catheter3,4,5. Bacteria associated with catheters characterized by their ability to form biofilm which is complex organic material forming of microorganisms growing in colonies within an extra-cellular mucopolysaccharide substance.Biofilm formation begins immediately after catheter insertion when organisms adhere to a consistingfilm of host proteins which forms along the catheter surface, both the interior and exterior catheter surfaces are involved6 .
Dextran is a bacterial homo-polysaccharide cationic polymer which the main chain consist of several α-glucans linked by α-(1-6) glycosidic bonds with different mount of branched linkages such as α-(1-2) , α-(1-3) and α-(1-4) linked as a single unite or lengthen side chain where the degree of branch depend on bacterial strain use for production7,8,9 .Certain lactic acid bacteria can produce dextran such as genera belong to Leuconostoc , Lactobacillus , Streptococcus, Pediococcus and Weissella10,11,12 . Dextran can be used as plasma/blood volume expanders, blood plasma substituent and drug delivery vehicle for a variety of drugs for its excellent biocompatibility, high biodegradability, wide availability and relatively low- cost modification through its reactive hydroxyl chemistry13.
Polyvinylpyrrolidone (PVP ) is define as a class of hydrophilic water-soluble polymer which attributing for electronegative groups of the carbonyl in the pyrrolidone structure that are able to form hydrogen bond with water 14, it is regarded as bulky, non-toxic,colorless , non-ionic , temperature-resistant, pH-stable , biocompatibility polymer with molecular formula of (C6H9NO)n and C=O, C-N , CH2 functional groups15,16,17 . PVP can be used in medicine field as solubilisers , stabilizers , medical additive, polymeric modifier , for sustaining drug and delivery 18. Attributing for its hygroscopicity , crosslinkability and low coefficient of friction , PVP can be used in catheter coating19.
The objective of this study was to determine the antibiofilm effect of biopolymer dextran produced by Leuconostocmesenteroides and their blends with gentamycin and PVP against pathogenic bacteria in catheters.
Materials and Methods
Microorganisms
An isolate of Leuconostoc mesenteroides ssp. mesenteroides was used for dextran production, which isolated from the fish intestine, then identified throughout cultural, microscopically and biochemical test according toGarvie20 and Vitek 2 system.
Ten isolates of bacteria isolated from catheters (collected from Iraqi males patients)included two isolates for each of Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus epidermidis and Staphylococcus aureuswere used forantibiofilmtest. These isolates were identified throughout cultural, microscopically, biochemical test according to the criteria established by Forbes21 and Vitek 2 system.
Productionof Dextran from L. mesenteroidesssp. mesenteroides
Aninoculum ofL. mesenteroidesssp. mesenteroides was prepared for dextran production by inoculating 10 ml of sucrose broth with a loop full of 24 h old culture of the isolate . After incubation for 24 h at 30oC, 1ml of this containing (9×108cfu/ml) was added to 100ml of the medium described bySarwatet al.22which contained (g/l): (sucrose 150 g, peptone 5.0 g , K2HPO4 15.0 , MnCl2.4H2O 0.01, yeast extract 5.0, NaCl 0.01 , CaCl2 0.05). Incubation was done at 30oC for 24 h23.
Precipitation and Purification of Dextran:
For dextran precipitation the equal volume of chilled ethanol was added to culture medium, shaken using the vortex, centrifuged at 8000 rpm for 20 min and the supernatant was decanted. This step was repeated twice23.The precipitated dextran was dried in oven approximately at 40oC for 45 minutes.
For dextran purification, the precipitated dextran was dissolved in distilled water then the dextran slurry that gained was precipitated with equal volume of chilled ethanol. This procedure of re-dissolving, precipitation and washing were repeated three times to eliminate cells debris22.Purified dextran was dried by using the electrical oven at 40oC for 45 minutes, and then dried dextran was calculated on dry weight basis.
Minimum Inhibitory Concentration (MIC) of Dextran, Gentamycin,PVP.
Minimum inhibitory concentration (MIC) values were determined against bacteria isolated from the catheters using broth dilution method as described byMorello et al24. Briefly, a stock solution of dextran from L.mesenteroidesssp. mesenteroides, gentamycin(Bioanalyse/Turkey) , PVP (Sino reagent /China) separately in
sterilized distilled water was diluted to concentrations ranging from (2-512)mg/ml , (2-512)µg/ml and (2-512)mg/ml respectively.
Antibiofilm Effect of Biopolymer Dextran - Gentamycin – PVP Blend in Micro titer Plate
The antibiofilm activity of biopolymerdextran produced by L. mesenteroidesssp. mesenteroides and blenders (gentamycin and PVP) against bacteria isolated from catheters were quantified using a plastic Microtiter plate as a primary method to detect the biopolymer and blenders abilities to coated surfaces and inhibition of biofilm formation, according to the procedure described by Ali 25 with modification , control wells full with 200µl of distilled water ,while other wells full with (200µl) subMIC of biopolymer, gentamycin , PVP alone and blends separately , the covered microtiter plate was sealed with parafilm during incubation at 37oC for 24 h , wells contents were decanted and washed with distilled water and dried for 15 min at room temperature , after drying (200µl) of bacterial suspension was added to the wells then sealed microtiter plate with parafilm and incubated at 37oC for 24 h . After incubation, wells contents were decanted then washed with distilled water. After drying at room temperature for 15 min 200µl of crystal violet (0.1%) was added tothe wells for 20 min . The stained wells were rinsed three times with distilled water, allowed to dry at room temperature for 15min then extracted with 200µl of 95% ethanol and the absorbance of each well was measured at 630nm using ELISA Reader. The inhibition of biofilm formation percentage of biopolymer, gentamycin, PVP and their blends was calculated as equation described by Namasivayamet al3 .
%Inhibition of biofilm formation =
Antibiofilm Effect of Biopolymer Dextran –Gentamycin – PVP Blend in Catheters
The effect of biopolymerdextran-gentamycin-PVP blend on biofilm formation of bacteria in catheters was examined according to method of Namasivayamet al3 with modification. Briefly, catheter was cut into pieces of 1.5cm and autoclaved, then the 1.5cm pieces were immersed separately in subMIC of biopolymer dextran, gentamycin, PVP , biopolymerdextran-gentamycin blend (1:1) , biopolymer dextran-PVP blend (1:1) and biopolymer dextran -gentamycin-PVP blend (1:1:1). A control catheter piece was without any coating treatment and then incubated at 37oC for 24 h in order to coating catheter pieces. After coating, the pieces were put on filter paper to removed solution and drying at 40oC. The dried pieces were immersedin 10 ml of nutrient broth that inoculated with E.coli and S.aureus separately, and then they incubated at 37oC for 24 h. After incubation, the broth was decanted then all coated and not coated catheter pieces were stained with (0.1) crystal violet for 30 min at room temperature. After staining, catheter pieces were washed with distilled water to remove excess stain and washed three times with 95% ethanol, then ethanol was collected for measuring the absorbance of each piece at 630nm using spectrophotometer and inhibition of biofilm formation percentage was calculated as equation described by Namasivayam et al3 .
Results and Discussion:
Minimum Inhibitory Concentration (MIC) of Dextran, Gentamycin, PVP.
The antibacterial activity of biopolymer dextran was determined on the basis of minimum inhibitory concentration (MIC) values. Results showed that the MIC of Biopolymer dextran was found to be 32mg/ml for E.coli , P.aeruginosa , S.aureus isolates , the MIC for P.mirabilis isolates was between (16 –32 ) mg/ml while in S.epidermidis was between (32 – 64)mg/ml.
The MIC of gentamycin was found to be 16 µg/ml for all bacterialisolates. The bactericidal activity of aminoglycosides such as gentamycin is dependent on the concentration. According to CLSI 26 , the MIC of gentamycin for most of Enterobacteriaceae such as E.coli , P.mirabilis and P.aeruginosa was ≥16 µg\ml as well as S.aureus and S.epidermidis .
The MIC of PVP was found to be 256 mg/ml for all bacterial isolates. Abdel-Aziz and Aeron 27 showed that the polymer was capable of inhibiting ≥ 99% of S.epidermidis , E.coli, and S.aureus . Oyanagiet al28
showed that 50% of bacteria died after treated with PVP. The presence of higher concentration of PVP improved membrane performances in the antibacterial activity29 .
Antibiofilm Effect of Biopolymer Dextran - Gentamycin – PVP Blend in Micro titer Plate
Antibiofilm effect of biopolymer dextran and blenders (gentamycin and PVP) against bacteria isolated from catheters was studied using micro titer plate. Results showed the ability of biopolymer dextran and its blenders to inhibit biofilm formation. Biopolymer dextran was recorded inhibition of biofilm formation for pathogenic bacteria isolated from the catheters with inhibition ratio ranged between (15-71) % and the maximum inhibition was against P.aeruginosa (Ps2), while gentamycin recorded biofilm inhibition between (3-79)% and the maximum inhibition was against S.epidermidis (Se2). In PVP it was between (39-70)% with maximum inhibition against S.epidermidis(Se2), while biopolymer dextran - gentamycin blend had biofilm inhibition ranged between (22-70)% and the maximum inhibition was against P.mirabilis (Pr2) , for dextran -PVP blend biofilm inhibition (23-65)% was recorded with maximum inhibition against P.mirabilis (Pr2) and in dextran-gentamycin-PVP blend , it was between (58-80)% with maximum inhibition against S.epidermidis (Se1) (Table 1).
From the results above, biopolymer dextran produced from L.mesenteroides ssp. mesenteroidesand its blenders were able to inhibit biofilm formation of gram positive and gram negative bacteria isolated fromcatheters.
Table(1):Antibiofilm effect of biopolymer dextran-gentamycin-PVP blend (Microtiter plate).
Bacterial isolates |
Biofilm inhibition % |
|||||
Dextran |
GM |
PVP |
Dex+GM |
Dex+PVP |
Dex+GM+PVP |
|
E.coli(E1) |
55 |
58 |
51 |
39 |
41 |
58 |
E.coli(E2) |
50 |
57 |
59 |
50 |
34 |
60 |
P.aeruginosa(Ps1) |
15 |
3 |
39 |
28 |
49 |
63 |
P.aeruginosa(Ps2) |
71 |
64 |
45 |
63 |
39 |
70 |
P.mirabilis(Pr1) |
55 |
42 |
54 |
51 |
42 |
55 |
P.mirabilis(Pr2) |
50 |
64 |
49 |
70 |
65 |
73 |
S.aureus(Sa 1) |
49 |
13 |
45 |
22 |
23 |
51 |
S.aureus(Sa 2) |
31 |
57 |
59 |
49 |
46 |
65 |
S.epidermidis(Se1) |
38 |
50 |
46 |
49 |
39 |
80 |
S.epidermidis(Se2) |
28 |
79 |
70 |
67 |
61 |
63 |
Dex: dextran, GM : gentamycin , PVP : polyvinylpyrrolidone
Antibiofilm Effect of Biopolymer Dextran –Gentamycin – PVP Blend in Catheters
The effect of biopolymer dextran produced by L. mesenteroides ssp. mesenteroides and blenders ( gentamycin and PVP) were studied to inhibit biofilm formation in the catheters. Results showed that biopolymer dextran was able to inhibit biofilm formation of pathogenic bacteria in catheters with biofilm inhibition 65% for E.coli(E 2) after incubated for 24 h and reached to 78% after incubated for 72 h , while against S.aureus(Sa 2) it was 57% after 24 h and reached to 58% after 72 h , while results showed that PVP have limited ability to inhibit biofilm formation of pathogenic bacteria on catheters , with 2% biofilm inhibition against E.coli(E 2) after 24 h and reached to 5% after 72 h , while against S.aureus(Sa 2) , was 15% after 24 h and reached to 20% after 72 h .
On other hand results showed that gentamycin was able to inhibit biofilm formation of pathogenic bacteria on catheters , where biofilm inhibition against E.coli(E 2) was 62% after 24 h but it was 46.9% after 72 h , while against S.aureus(Sa 2) was 59% after 24 h but it was 55% after incubated for 72 h .Our results also showed that biopolymer dextran – Gentamycin blend was able to inhibit biofilm formation of pathogenic bacteria on catheters ,where against E.coli (E 2) was 69% after 24 h but it was 85% after 72 h while against S.aureus(Sa 2) was 52% after 24 h but it was 75% after incubated for 72 h .Also results showed that biopolymer dextran – PVP blend was able to inhibit biofilm formation of pathogenic bacteria on catheters ,where against E.coli (E 2) was 68% after 24 h but it was 43% after 72 h while against S.aureus(Sa 2) was 59%
after 24 h but it was 50% after incubated for 72 h .In dextran - gentamycin - PVP blenders, results showed ability of blend to inhibit biofilm formation of pathogenic bacteria on catheters, where biofilm inhibition against E.coli (Sa 2) was 71% after 24 h but it was 90 % after 72 h , while against S.aureus(Sa 2) was 63% after 24 h but it was 81% after incubated for 72 h (Table2), (Figure1) .
Biopolymer dextran as an anti-adhesive polymer that worked as antibiofilm30where dextran with Iron oxide nanoparticle can act as antibiofilm against E.faecalis and P.aeruginosa31 . Exopolysaccharide hadantibiofilm activity against pathogenic bacteria such as EPS that produced by Lactobacillus fermentum32 . In another study, Wu et al33 observed antibiofilm activity of exopolysaccharide extracted from marine bacteria against P.aeruginosa while Sayemet al34 reported novel exopolysaccharide isolate from Bacillus licheniformis with the antibiofilm activity.
Moreover,Kanmani et al35 used exopolysaccharide extracted from E.faecium as antibiofilm against Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus and Pseudomonas aeruginosa . Antibiofilm effect of polysaccharide (as sugar polymers) are explicated to their ability to alter the physical characteristics of bacterial cells or abiotic surfaces , act as signaling molecules that affect the gene expression of bacteria or by inhibiting carbohydrate- protein interactions , thereby interfering with adhesion27 .
In PVP , studies on antibiofilm activity of PVP - Fe3O2 combination on the surface of a medical device like the catheter showed that PVP - Fe3O2 had the ability to inhibited biofilm formation against S.aureus and P.aeruginosa36 . Dispersion forces between the poly chain and the bacterial cells prevent bacteria from binding to the surface and initiating biofilm growth. Modification of the surface change of polymers has also proven to be an effective means of biofilm prevention27 .
Other studies used gentamycin with a biodegradable polymer to prevent biofilm formation by S.aureus on metallic surgical implants37 while Lin et al38 observed the ability of gentamycin loaded titania nanotube to inhibited biofilm formation . Such studies, Machado et al39 revealed the ability of P.aeruginosa to form biofilm under pressure of antibiotic like ciprofloxacin and gentamycin . However, none of the antibiotics could eradicate biofilm completely. Henry-stanleyet al40 demonstrated the ability of aminoglycoside group (gentamycin) to inhibited of S.aureus biofilm formation while Mu et al41 used chitosan with the gentamycin as antibiofilm to improved antibiotic penetration .
The essential challenge for restore functions of catheters and prevent trauma, disease or ageing is by producing a coating with an antibacterial activity which resist for a long period of time in the device , not eliminated by contact with urine and have a board spectrum activity without spread bacterial resistance . A biomaterial coating is the most biocompatibility agent can decrease biofilm formation and more comfortable for the patient when catheter inserted in the body or in urethra42 . Fisher et al43 suggested biomaterial modification of urinary catheters with antimicrobials activity where polymer of three antibiotics combination of rifampicin, sparfloxacin and triclosan were used to prevent colonization by common uropathogenic P.mirabilis, S. aureus and E. coli to give long-term broad spectrum antibiofilm activity while Islas et al44 used poly (vinyl chloride) (PVC) urinary catheters grafted with polymer to preventing biofilm formation in catheters and avoid bacteria adhesion. Also, ciprofloxacin-loaded catheters inhibited the growth of E. coli and S. aureus surroundings the catheter and prevented bacteria adhesion .
Table(2):Antibiofilm effect of biopolymer dextran-gentamycin-PVP blend in catheters.
Bacterial isolates |
Incubation time (h) |
Biofilm inhibition (%) |
|||||
Dextran |
GM |
PVP |
DEX.GM |
DEX.PVP |
DEX.GM. PVP |
||
Escherichia coli (E2) |
24 |
65 |
62 |
2 |
69 |
68 |
71 |
48 |
71 |
57 |
3 |
76 |
53 |
78 |
|
72 |
78 |
47 |
5 |
85 |
43 |
90 |
|
Staphylococcus aureus(Sa2) |
24 |
57 |
59 |
15 |
52 |
59 |
63 |
48 |
57 |
58 |
17 |
63 |
55 |
74 |
|
72 |
58 |
55 |
20 |
75 |
50 |
81 |
Dex: dextran, GM : gentamycin , PVP : polyvinylpyrrolidone
Coated catheter
Control catheter
Figure(1):Antibiofilm effect of biopolymer dextran – gentamycin – PVP blend against E.coli in catheter after 72 h .
Conclusion
In conclusion, the biopolymer dextran- gentamycin – PVP blends had antibiofilm properties against pathogenic bacteria isolated from catheters. Also had the potential to be used as antibiofilmcoating for catheters.
Reference
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