Battling Biofilm Forming Nosocomial Pathogens Using Chitosan and Pluronic F127

Biofilm represents a potential strut in bacterial treatment failure. It has a dual action; it affords microbial resistance against antibiotics and facilitate transmission of pathogenic bacteria. Nosocomial bacteria pose a serious problem in healthcare units; it prolongs patient hospital stay and increases the mortality rates beside other awful economical effect. This study was planned for targeting nosocomial bacterial biofilm using natural and biologically safe compounds like Chitosan and/or Pluronic F127. Ninety-five isolates were recovered from 107 nosocomial clinical samples. Different bacterial and fungal species were detected, from which Klebsiella pneumonia (23%), Pseudomonas aeruginosa (19%), Acinetobacter baumannii (18%) and E.coli (17%) were the predominate organisms. Pseudomonas aeruginosa, Acinetobacter baumanni and Klebsiella pneumonia were the abundant antibiotic resistant strains with multi-resistance pattern of 72%, 65% and 59%, respectively. A significant percentage of these isolates were strong biofilm forming. Herein, we investigate the effect of Chitosan and Pluronic F127 alone and in combination with each other against biofilm production. Chitosan show variable degree of biofilm inhibition, while Pluronic F127 was able to retard biofilm formation by 80% to 90% in most strain. There is no significant difference (P< 0.05) between Pluronic F127 alone and its effect in combination with Chitosan.

activity at lower concentration. Molecular weight and acetylation degree affect its potency as antimicrobial agent 15 . Being as positively charged cations, chitosan can act in 3 different ways: by interaction with negatively charged microbial cells; interact with microbial DNA; or chelating important metals required for metalloprotein enzymes 16 .
Pluronic F127 is a synthetic non-ionic surfactant with amphiphilic properties. It is a copolymer of hydrophilic poly(ethylene oxide) and hydrophobic poly(propylene oxide). Due to their amphiphilic characters, Pluronic has an excellent surfactant property 17 . Combination of chitosan and pluronic acid were mixed together in a nanoparticle form for delivery of anticancer drugs with less side effect 18,19 . The combination is also used for preparation of different pharmaceutical dosage forms 20, 21 The aim of this study is to evaluate efficacy of chitosan and pluronic F127 alone and in combination with each other against biofilm forming nosocomial pathogens as a new tactic to retard their transmission and resistance.

MATerIAl AND MeTHOD Sample Isolation
Different biological samples (urine, sputum, endotracheal secretion and blood) were collected from 107 patients between May 2017 and June 2018. All patients admitted to hospital for at least 3 days without previous signs or symptoms of previous infection. All samples were isolated from patients in ICU and neonatal ICU following ethical consideration.
Strains were isolated and purified using different types of media (Blood agar and MacConkey agar). All isolates were stored in glycerol broth at -80°C till further analysis.

Biofilm Formation Assay of Isolated Pathogens
A previously isolated pure colony was resuspended in 5 mL of tryptone soya broth supplemented with 1% glucose, incubated at 37°C or 30°C for 48 hrs for bacteria and Candida, respectively. Twenty microliters of overnight culture were diluted in 180 µL of the above media in 96 well sterile microplate and incubated at 37°C or 30°C for 48 hrs. After incubation the growth was discarded, and the plates were washed three times with phosphate buffered saline pH 7.5 to remove non-adherent cells. The plates were dried in the oven at 65°C and stained with 200 µL of 1% crystal violet solution for 15 min. the plates were washed gently under running water and dried. A solution of 1% acetic acid is used to retain adsorbed C.V stain for 15 min. and measured spectrophotometrically at 600 nm by microplate reader (Tecan SunRise/ USA). The strains were classified as weak, moderate or strong biofilm forming bacteria according to classification of Stepanovic et al. 23 The experiments were done with six replicates in three independent experiments.

effect of Chitosan and Pluronic Acid on Biofilm Formation
Chitosan was dissolved in 1% acetic acid solution to a final concentration of 10 mg/mL. The solution was stirred overnight at 50°C to ensure complete dissolution, pH was raised to 5.8-6.0 by 1 N NaOH and sterilized by 0.2µm microbial filter (Sartorius, Germany). Pluronic F127 was dissolved at a concentration of 10 mg/mL in distilled water at pH of 7±0.2. Bacterial overnight culture was diluted in TSB amended with 1% glucose to a concentration of 10 8 CFU/mL containing different concentrations of Chitosan and/or Pluronic F127 (5, 2.5, 1.25, 0.625 mg/ml). In a flat bottom microtiter plate, a 200 µL of the above solution was added and incubated at 37°C or 30°C for 48 hr. Extent of biofilm inhibition occurred by Chitosan and/or Pluronic F127 is detected by C.V method. Percentage of inhibition is calculated by the following equation by Pierce et al. 24 Inhibition %= 100-(OD sample /OD control ) x 100 Scanning electron Microscope for Biofilm Inhibition Assay Ten microliter of 0.5 MacFarland solution of diluted overnight bacterial culture was placed in the center of Sterile Millipore membrane filter (0.22 µm, 47 mm diameter, mixed cellulose esters (MCE) membrane) (Merck, USA). The filter was loaded on tryptone soya agar plates (TSA) amended with 2.5 mg/mL of Chitosan, Pluronic F127 and combination of Chitosan and Pluronic F127. The plates were incubated at 37°C for 24 hr. The membrane filters were gently removed, fixed in 3% glutaraldehyde for 30 min. The filters were washed 3 times with PBS each for 10 min. The membranes were gradually dehydrated with gradient conc of ethyl alcohol (50%, 60%, 70%, 80%, 90%, 95%, 100%), then final chemical dehydration with hexamethyldisilazane. The coupons were coated with gold, and then examined with JSM-6510 (JEOL, Japan) at a voltage of 30 kV and magnifications at x5000 to ×15000 25 .

Statistical Analysis
Data were expressed as mean ± standard error of mean (SEM). Statistical analysis was performed using statistical package for social sciences (SPSS) computer software (version 22), IBM software, USA. One-way ANOVA test was used to evaluate significance between groups followed by tukey posthoc analysis for pairwise analysis. Differences were considered statistically significant at p<0.05.

Bacterial Isolation and Identification
Out of 107 clinical samples taken, 95 samples were positive for bacterial or fungal culture. Large number of isolated strains were found in sputum (30 strains) and endotracheal intubation (28 strains). Urine samples were positive for 27 specimens while blood gives only 10 isolates (Table 1).
The top four species were Klebsiella pneumoniae (23%), Pseudomonas aeruginosa (19%), Acinetobacter baumannii (18%) and E.coli (17%). Gram +ve bacteria also present Journal of Pure and Applied Microbiology in a significant percentage represented by Staphylococcus aureus and Staphylococcus haemolyticus with 8% and 4% respectively. Other Gram-negative bacteria like Proteus spp. and Enterobacter spp. were found in a small frequency (3% and 2% consequently). Fungal infection with Candida albicans and Candida tropicalis were also reported with a lower incidence at 3 and 2% respectively. (Table 1)

Antibiotic Sensitivity Testing
All Gram-positive strains were sensitive to Vancomycin, while all Gram-negative strains were sensitive to Colistin. Ten isolates (45%) of Klebsiella, seven isolates (41%) of Acinetobacter and six isolates (33%) of Pseudomonas show resistance against all tested antibiotics. Four strains of E.coli and one isolate of Staphylococcus haemolyticus, Enterobacter spp., and Proteus spp.
were resistant to nine of tested antibiotics. Overall, resistance to the Piperacillin/tazobactam and Imipenem were found to be much lower. (Table 2)

Biofilm Formation Assay of Isolated Pathogen
Thirty-five isolates show no biofilm activity, while 26 and 24 isolates show weak and moderate biofilm forming ability, respectively. Only 10 isolates were strong biofilm producer and they were distributed as one strain of St. haemolyticus, one strain Candid tropicalis, three strains of Klebsiella pneumonia, two strains of Acinetobacter baumannii and three strains of Pseudomonas aeruginosa. (Fig. 1).

effect of Chitosan and Pluronic F127 on Biofilm Formation
All isolated strains were significantly reduced to different levels by chitosan at the concentration used 2.5 mg/mL. The most affected strain was the extensively drug resistant Pseudomonas aeruginosa (XDR) strain, it was decreased by 81% of initial biofilm formed in planktonic cells. Multi-and extensive drug resistant Klebsiella pneumonia strains (MDR and XDR) and Acinetobacter baumannii were inhibited by 25-35%. Also, chitosan diminished biofilm of Pseudomonas aeruginosa (MDR), Staphylococcus haemolyticus and Candida albicans by 33%, 51% and 58% respectively. (Fig. 2) Pluronic F127 has a more potent effect than Chitosan in prevention of biofilm creation at concentration used 1.25 mg/mL. It inhibits all Pseudomonas and Klebsiella spp. by more than 80%. It also prohibits biofilm production in other strains of Staphylococcus haemolyticus, Acinetobacter baumannii and Candida albicans by 69%, 43% and 59% respectively. Statistical analysis shows significant difference (P<0.05) in biofilm inhibition between Pluronic F127 and Chitosan in Klebsiella pneumonia (XDR, MDR) and Pseudomonas aeruginosa (MDR). Other strains show no significant difference between Pluronic F127 and Chitosan. overall, all strains show no significant difference between Pluronic F127 and Mixture of both materials. (Fig. 2)   Fig. 1. Biofilm forming ability of clinical isolates recovered from different nosocomial clinical samples.

electron Microscopic Image of Bacterial Strains
Biofilm inhibition by chitosan and/or pluronic acid were also confirmed by imaging with scanning electron microscope (SEM). Untreated strains show a high percentage of exopolysaccharide matrix with increased aggregation of cells in thick multicellular pattern. While, treated cells with chitosan and/or pluronic acid show a well isolated microcolonies with limited or no exopolysaccharide materials formed. (Fig. 3,4)

DISCUSSION
Most nosocomial infections are attributed to organisms with considerable degree of antibiotic resistance. This leads to increased demand on discovering new types of antibiotics with decreased resistance. Targeting bacterial biofilm is another strategy to reduce their transmission and increase efficacy of antibiotics.
For this work, Chitosan has a variable inhibitory effect on biofilm formation regarding different bacterial species. These findings are in agreement with Polyudova et al. who found that Chitosan has a fourfold inhibition of Mycobacterium smegmatis biofilm while has a minimal effect on E.coli strains, he refer his foundation to the increase in the hydrophobicity of attachment surfaces that will decrease the effect of chitosan as a biofilm inhibitor 39 . Puga and his colleagues suggest that environmental stress leads to increased tolerance to Chitosan effect 40 . Also, molecular weight and acetylation degree may control the effectiveness of Chitosan 41 .
Pluronic F127 shows potent antibiofilm activity at low concentration (1.25 mg/mL), its activity reaches up to 80% inhibition for strong biofilm producing strains. Treter et al. show similar effect of Pluronic F127 on Staphylococcus epidermidis, it inhibits 90% of biofilm formation 42 . Although, combination of Pluronic with Chitosan has a similar effect like Pluronic alone, but Pluronic can increase release, solubility and bioavailability of Chitosan. Alvarado-Gomez et al. reported synergistic activity of Pluronic F127 and silver nanoparticles in hydrogel form against biofilm forming Pseudomonas spp and Staphylococcus spp 43 . Another study by Manaspon et al. reported increased cytotoxic activity of doxorubicin against breast cancer cells using folate-conjugated pluronic F127/chitosan core-shell nanoparticles 18 . From our work and previous studies, we conclude that Pluronic F 127 can increase effect of Chitosan suggesting their use in combination at low concentration level with high efficacy against biofilm formation.

CONClUSION
Nosocomial infection represents a substantial health problem. A significant number of nosocomial isolates were moderate to strong biofilm producers. Despite of finding a new effective antibiotic for treatment of highly resistant organisms, inhibition of transmission can represent a new effective approach. Chitosan and Pluronic F127 are safe biocompatible agents used in different medical formulation. They show a potential degree of biofilm inhibition. They could be used as an alternative source for inhibition of bacterial resistance and transmission.

ACKNOWleDGMeNTS
We acknowledge Dr. Mohammed Ragab (Pharmacology Department, Faculty of Pharmacy, Beni-Suef University) for performing statistical analysis using SPSS software.

AUTHOrS' CONTrIBUTION
AFA and WGH design the experiment; DE, AFA performed the experiments; WGH, AFA, OMS analyzed the data; AFA, OMS write the original draft. All authors read and approved the manuscript.

FUNDING
None.