Antibiogram of chronic ulcers from
Healthy human skin has beneficial microflora and many pathogens causing infections. Staphylococcus aureus is the most prevalent and can have multiresistance to antibiotics. Chitosan is a polysaccharide composed of glucosamine and N-acetyl-D-glucosamine, which is biodegradable and has antimicrobial activity. As part of a national scientific research project for the development and application of biomaterials, we decided to study the effect of different membranes based on chitosan against strains of S. aureus isolated from infected ulcers. The study found that seven of nine strains of S. aureus are sensitive to rifampin and the least eight of nine strains were multiresistant to more than ten antibiotics. All chitosan-based membranes confirm its antimicrobial effect on direct contact with an increase in its diameter. The contact area of the membranes is increased according to the concentration of chitosan. The highest average area increase was the chitosan membranes with honey and glycerin, 88.32%. Chitosan membranes have shown their effectiveness against S. aureus strains of clinical origin. Thus, these materials can be applied for the treatment of chronic ulcers without toxic hazards and resistance caused by antibiotics.
- antimicrobial membranes
Chitosan is a cationic linear polysaccharide composed of glucosamine and N-acetyl glucosamine units linked by β (1-4) glucoside bonds . This polymer is biodegradable and has antimicrobial activity against
The skin surface is characterized by a slightly acidic pH that favors the development of some bacteria . However, in an open wound, within the first 24–48 h can be found
Skin infections affect the epidermis, subcutaneous tissue, and muscle. Some of them require hospital admission for antimicrobial or surgical treatment. Depending on their severity, they have been associated with increased hospital stays and medical costs because traditional antibiotic treatments require long periods . An alternative to antibiotics is curing materials with bioactive components. These dressings should ideally maintain a moist environment, act as antimicrobials to prevent secondary infections, remove exudate, and promote tissue regeneration [9, 10] One group evaluated the antimicrobial action of cotton textile impregnated with chitosan against bacteria isolated from the skin and found that chitosans of low- and high-molecular weight showed effective inhibition of
Chitosan was obtained by thermo-alkaline hydrolysis of chitin, which was recovered by lactic fermentation of shrimp waste. The chitin was demineralized (0.1 M HCl at 25°C for 4 h) and deproteinized (4.5% NaOH at 65°C for 4 h). Afterward, purified chitin was deacetylated (40% NaOH at 110°C for 2 h) to obtain chitosan, according to reported methodologies . Chitosan was washed until a neutral pH was reached and dried at 50°C. The purity of the chitosan was verified based on its moisture and ash content by method given in reference .
Degree of deacetylation of chitosan was determined by a spectrophotometric method reported by Liu et al. . Briefly, two standard solutions were prepared; D-glucosamine (7.49 mM) and N-acetylglucosamine (0.49 mM), from these, working solutions were prepared to obtain a 12-point line of different concentrations. The absorbance of the standard solutions and samples were read at 201 nm in a UV-Vis spectrophotometer (Genesys 10 UV, Madison, IA, USA). Finally, the degree of deacetylation was calculated with the following equation, %DD = (161.1 ×
The molecular weight of chitosan was determined as proposed by Solis et al. , based on the intrinsic viscosity, according to Mark-Houwink´s equation
Chitosan was identified by Fourier Transform Infrared Spectroscopy (FTIR) according to the methodology given in reference , with some modifications. The spectral resolution was of 4 cm−1 with 64 scans in a range of 600–4000 cm−1 using a Thermo Scientific (Nicolet5s, Madison, IA, USA) infrared spectrometer.
2.2. Chitosan membranes
Six chitosan based formulations using 1% acetic acid were prepared. Three pure chitosan solutions (1, 2, and 3%), another of 2% chitosan with glycerin (five drops per 100 ml) and two more solutions of 2% chitosan using honey (95:5, v/v) with and without glycerin. For the last two solutions, honey was diluted in distilled water (80:20, v/v) and homogenized by stirring.
All chitosan membranes were prepared by solvent evaporation. For this, solutions of each formulation were poured into polypropylene plates (10 ml in each mold) and dried at 40°C for 24 h in an oven (Felisa, Zapopan, Jalisco, Mexico). Lastly, the films were removed from the mold and stored in sterile plastic bags until use.
2.3. Collection and conservation of the sample
Between June and December 2015, a descriptive and cross-sectional study was conducted in order to evaluate the effect of chitosan membranes against
The samples were obtained from the center of the ulcer by a medical epidemiologist. For this, a Stuart medium swab collection system (COPAN Transystem, Brescia, Italy) was used. The labeled samples were transported in a container to a certified microbiology laboratory for processing within the same hour.
2.4. Phenotypic identification and sensitivity tests
For microbial isolation and identification, the samples were inoculated by cross-streaking on MacConkey agar for Gram-negative bacterium and trypticase soy agar (TSA) for culture collection. Mannitol salt agar was used for Gram-positive bacterium and Biggy agar for fungi inoculated by streaking. Next, the plates were incubated at 37°C for bacteria and 30°C for fungi for 24 h. Later, macroscopic characteristics of colony-forming units (CFU) were analyzed.
For the identification of microorganisms, a broth microdilution method was used, with an inoculation Prompt™ system precision wand. Isolated colonies were emulsified in Prompt™ innoculation bottles for an equivalent concentration of 0.08 with the McFarland standard. Next, 100 μl per well were deposited in the SIEMENS microplates from a MicroScan RENOK panel which was incubated at 37°C for 24 h. Specifically, type 33 plates (B1017-211) were used for Gram-positive bacteria and type 44 (B1017-305) for Gram-negative bacteria. Later, each plate was read for the identification of studied microorganisms using a LabPro Command Center software. Antibiotic sensitivity was performed simultaneously with phenotypic identification using a RENOK MicroScan panel. The MIC (Minimum Inhibitory Concentration) was determined according to the CLSI (Clinical and Laboratory Standards Institute) criteria for each antibiotic. The studied antibiotics were oxacillin, gentamicin, tetracycline, daptomycin, ampicillin, erythromycin, penicillin, nitrofurantoin, vancomycin, levofloxacin, moxifloxacin, ciprofloxacin, linezolid, ceftriaxone, Sinercid, clindamycin, rifampin, amoxicillin/clavulanate k, trimethoprim/sulfamethoxazole, and ampicillin/sulbactam.
2.5. Sensitivity tests with chitosan membranes
The sensitivity test for
Chitosan based membranes were cut into 16 mm diameter discs and placed in triplicate on the inoculated agar with sterile forceps, ensuring direct contact between both surfaces. Each agar also included two blank controls that consisted of Whatman # 1 paper; one was impregnated with 1% acetic acid and the other with 0.9% sodium chloride. Plates were incubated at 37°C for 24 h. Lastly, the antimicrobial effect of chitosan was evident by observing the existence of inhibition zones below the membranes and inhibition halos around each membrane.
3. Results and discussion
3.1. Chitosan characterization
The ash content of chitosan is an indication of its purity. For chitosan with 9.42 ± 0.07% moisture, the average ash contents are 0.37 ± 0.02%. The range of ash content is 0.08% for langoustine obtained chitosan  and 4.0% to crab obtained chitosan , whereas for chitosan obtained from shrimp, the range is from 0.070  to 0.832% . Variations in ash content are due to the location of origin, as well as the purification and thermos-alkaline hydrolysis of chitin.
The molecular weight of chitosan determines its functional properties and its antimicrobial activity. This study found that the average molecular weight of chitosan was 119.48 kDa, estimated based on the intrinsic viscosity. With this value, chitosan can be classified as a low molecular weight (50–190 kDa) material. Therefore, the molecular weight is a reflection of the process conditions used to obtain chitosan from the purification and thermo-alkaline deacetylation of chitin. Other investigations have reported molecular weights for shrimp obtained chitosan of 136 , 180 , and 1260 kDa . While the values reported for chitosan obtained from crab shells vary from 1240  to 483 kDa . For fungal chitosan, reports show low molecular weight, from 41  to 110–150 kDa . Davoodbasha et al.  conducted antimicrobial tests with commercial chitosan 100–300 kDa. Hernández-Ochoa et al.  also studied the antimicrobial activity of commercial chitosan with different molecular weight (low: 50–190 kDa, medium: 190–310 kDa and high: 310–375 kDa) finding satisfactory results with the lower molecular weight.
For the degree of deacetylation of chitosan, an average value of 84.59 ± 0.87% (
The FTIR technique made it possible to identify the functional groups in the chitosan molecule (Figure 1). At 881.96 cm−1, a characteristic band of the stretching of the glycosidic bond can be observed; also at 1055.62 and 1028.60 cm−1 the stretching of C─O can be seen. It is possible to observe the bending band N─H of the primary amide formed during deacetylation at 1545.72 cm−1 and the amine group at 1626.76 cm−1. At the position of 2884.84 cm −1 the stretching of C─H and at 2962.02 cm−1 the tense vibration from group C─H can be observed. Ultimately, OH and N─H were identified at 3274.61 and 3359.51 cm−1, respectively. These results are similar to those reports given by references [32, 34]. As reported by Shigemasa et al. , errors in band intensity can occurred at positions 1640 and 3450 cm−1 due to the effect of water absorption.
3.2. Preparation of chitosan membranes
Dissolved chitosan is capable of forming a membrane due to the evaporation of the solvent, as well as forming intra and intermolecular hydrogen bonds between the chitosan polymer chains . Chitosan membranes were prepared using pure chitosan and a mix of chitosan with glycerin or honey. All membranes were transparent, uniform, and smooth at the surface and with hard texture when chitosan concentration was increased. Pure chitosan membranes show a very pale yellow color and membranes containing honey show a brown tone. All membranes were easily removed from the polyethylene plates with 9.5 cm in diameter, and thickness depending on the concentration of chitosan, found in the range of 0.0131 ± 0.0021 and 0.0339 ± 0.0034 mm for 1 % and 3 %, respectively.
3.3. Phenotypic identification of microorganisms
From patients hospitalized for various reasons in a regional hospital located in northwestern Mexico, 23 infected skin ulcers derived from toes, sacral parts, coccyx, arm, and leg stumps were analyzed. The age range of the patients was from 43 to 96 years. Among the isolated and identified strains were
|Oxacillin||R ˃2||S < 0.25||S < 0.25||R ˃ 2||R ˃ 2||R ˃ 2||R ˃ 2||R ˃ 2||R ˃ 2|
|Gentamicin||S < 4||S < 4||S < 4||R ˃ 8||R ˃ 8||S < 4||R ˃ 8||S < 4||S < 4|
|Tetracycline||S < 4||S < 4||S < 4||R ˃ 8||R ˃ 8||R ˃ 8||S < 4||S < 4||S < 4|
|Daptomycin||R ˃ 4||R ˃4||R ˃4||R ˃ 4||R ˃ 4||R ˃ 4||S < 0.5||R ˃ 4||S < 0.5|
|Ampicillin||R ˃ 8||R ˃ 8||S < 2||R ˃ 8||R ˃ 8||R ˃ 8||R ˃ 8||R ˃ 8||R ˃ 8|
|Erythromycin||R ˃ 4||R ˃4||R ˃4||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4|
|Penicillin||S < 0.03||R ˃ 8||R ˃ 8||R ˃ 8||R ˃ 8||R ˃ 8||R ˃ 8||R ˃ 8||R ˃ 8|
|Nitrofurantoin||R ˃ 64||S < 32||S < 32||S< 32||R ˃ 64||R ˃ 64||S < 32||S < 32||S < 32|
|Vancomycin||S < 0.25||S < 0.25||R ˃ 16||S < 0.25||S < 32||R ˃ 16||R ˃ 16||R ˃ 16||R ˃ 16|
|Levofloxacin||R ˃ 4||S < 1||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4|
|Moxifloxacin||R ˃ 4||S < 0.5||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4|
|Ciprofloxacin||R ˃ 2||S < 1||R ˃ 4||R ˃ 2||R ˃ 2||R ˃ 2||R ˃ 2||R ˃ 2||R ˃ 2|
|Linezolid||S < 1||S < 1||R ˃ 4||R ˃ 4||R ˃ 4||S < 1||S < 1||R ˃ 4||R ˃ 4|
|Ceftriaxone||R ˃ 32||S < 8||S < 8||S < 8||R ˃ 32||R ˃ 32||R ˃ 32||S < 8||R ˃ 32|
|Synercid||S < 0.5||S < 0.5||R ˃ 2||R ˃ 2||R ˃ 2||S < 0.5||S < 0.5||S < 0.5||S < 0.5|
|Clindamycin||R ˃ 4||S < 0.5||S < 0.5||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4||R ˃ 4|
|Rifampin||S < 1||S < 1||S < 1||R ˃ 2||R ˃ 2||S < 1||S < 1||S < 1||S < 1|
|Amoxicillin/K clauvulanato||R ˃ 4/2||S < 4/2||R ˃ 4/2||R ˃ 4/2||R ˃ 4/2||R ˃ 4/2||R ˃ 4/2||R ˃ 4/2||R ˃ 4/2|
|Trimethoprim/sulfamethoxazole||S < 0.5/9.5||S < 0.5/9.5||R ˃ 2/38||R ˃ 2/38||R ˃ 2/38||S < 0.5/9.5||R ˃ 2/38||R ˃ 2/38||S < 0.5/9.5|
|Ampicillin/sulbactam||R ˃ 16/8||R ˃ 16/8||S < 8/4||R ˃ 16/8||R ˃ 16/8||S < 8/4||S < 8/4||S < 8/4||S < 8/4|
S. aureus sensitivity to antibiotics
Table 1 shows the minimum inhibitory “
Figure 3 shows that most strains are resistant to the most commonly used antibiotics. All isolates were resistant to erythromycin. Only one of the nine strains showed sensitivity toward ampicillin, penicillin, levofloxacin, ciprofloxacin, and amoxicillin.
The most commonly used antibiotics for
S. aureus sensitive chitosan membranes
In a study by Hernández-Ochoa et al. , with
The antimicrobial properties of chitosan mainly depend on the degree of deacetylation and molecular weight, as well as pH and ionic strength of the medium . El-tahlawy et al.  and Hosseinnejad and Jafari  reported that low molecular weight chitosan can penetrate into the cell and inhibits mRNA and protein synthesis. Chitosan oligomers have higher antimicrobial effect due to their shorter chain and free amino groups from D-glucosamine . Similarly, Champer et al.  reported that the amount of free amino groups influence the antibacterial properties of chitosan. Likewise, Wang et al.  state that all bacteria possess negative charges; therefore, they are easily captured by the protonated amine groups of chitosan and lose their reproductive functions and bioactivity. Acetic acid has an effect on the solubility of the polymer and on the protonation of the amino groups.
Kim et al.  evaluated the antimicrobial activity of chitosan membranes of different molecular weights with
Additionally, it was observed that the membranes tend to increase their diameter by staying in contact with the agar, thus increasing the antimicrobial effect by contact. Table 2 presents the increase in diameter and area of the membranes at the end of agar diffusion assay. For pure chitosan membranes, it was found that an increase in size is directly proportional to its concentration. It was also observed that the addition of glycerin or honey increases the size of the chitosan membranes by 2%. The 2% chitosan membranes mixed with honey and glycerin showed the greatest increase, however, bacterial growth was observed.
|Membranes||Increased radius (mm)a||Increase in contact area (%)b|
|Qo 1%||9.01 ± 4.97||20.03 ± 14.90|
|Qo 2%||9.39 ± 6.48||30.61 ± 17.33|
|Qo 3%||9.70 ± 9.58||39.80 ± 25.80|
|Qo 2% + gly||9.74 ± 6.34||40.34 ± 17.38|
|Qo 2% + miel||11.20 ± 4.65||65.38 ± 20.29|
|Qo 2% + miel + gly||11.20 ± 4.53||88.32 ± 21.73|
Vlacha et al.  reported that free hydroxyl groups from chitosan interact with the moist atmosphere increasing the diameter of the membrane. Also, Zamora-Mora et al.  mentioned that pure chitosan membranes show a higher water holding capacity due to the hydrophilicity of the chitosan. According to Estrada et al. , honey potentiates the antimicrobial activity of chitosan due to chemical action of its components; hydrogen peroxide, organic acids and flavonoids, nectar, pollen, and propolis. For Grade et al. , plasticizers provide flexibility to the membranes, but weaken the intermolecular forces and cause the penetration of water through the membrane increasing its size .
Different types of chitosan-based membranes mixed with glycerol and honey were developed and characterized. These membranes showed antimicrobial activity against
AA Escárcega-Galaz gratefully acknowledges the Consejo Nacional de Ciencia y Tecnología (CONACYT) by PhD scholarship: 417707. This research was funded by the Instituto Tecnológico de Sonora (Project PROFAPI-00471) and by the Consejo Nacional de Ciencia y Tecnología (PDCPN2014: 248160).
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