Nova Biotechnol Chim (2021) 21(1): e806 DOI: 10.36547/nbc.806 1 Nova Biotechnologica et Chimica Effect of chitosan membranes against gram-negative bacteria isolated from cutaneous ulcers Jaime López-Cervantes 1 , Ana A. Escárcega-Galaz 1 , Dalia I. Sánchez-Machado 1, , Benjamín Ramírez-Wong 2 , Ramssel D. Valenzuela-Rojo 1 , Olga N. Campas-Baypoli 1 , Ernesto U. Cantú-Soto 1 , Carlos U. Mendivil-Gastelum 1 1 Instituto Tecnologico de Sonora, Ciudad Obregon, MX-85000 Sonora, Mexico 2 Universidad de Sonora, Hermosillo, MX-83000, Sonora, Mexico  Corresponding author: dalia.sanchez@itson.edu.mx Article info Article history: Received: 23 rd December 2020 Accepted: 7 th May 2021 Keywords: Antimicrobial agent Chitosan Escherichia coli Gram-negative Skin ulcers In vitro Membranes Antibiotic Antibiotic sensitivity Abstract The objective of this study was to evaluate the in vitro effectiveness of membranes developed with pure chitosan and chitosan in a mixture with glycerol-honey against gram-negative bacteria isolated from skin ulcers. The membranes were prepared by the solvent evaporation technique. The identification and antibiotic sensitivity of microorganisms were determined in microplates, and in vitro tests were developed by the agar diffusion technique. The most frequently isolated microorganism was Escherichia coli with 43.75 % prevalence. All membranes showed antimicrobial effects by direct contact against Proteus mirabilis, Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae and Morganella morganii. Antibiograms showed that most of these microorganisms are multi- resistant to antibiotics. All of this suggests that chitosan-based membranes are a safe alternative for the treatment of infected cutaneous ulcers compared to traditional antibiotics. The outcomes of this study confirm that membranes made of a biodegradable polymer, such as chitosan have activity against multidrug-resistant gram-negative bacteria that grow in infected skin ulcers.  University of SS. Cyril and Methodius in Trnava Introduction Chronic wounds cause severe financial problems due to the medical attention they require. These types of wounds are characterized by slow healing, or they may never heal, which generates physical and emotional stress to the patient. The degree of infection always depends on the species and concentration of the microorganism, as well as the response of the host (Akuzy et al. 2018). According to data reported by Davies et al. (2007), chronic wounds on the skin affect approximately 3 % of people over 60 years of age; additionally, these types of wounds maintain polymicrobial biota. The microbial biota found in chronic wounds includes Staphylococcus, Streptococcus, Pseudo- monas and coliforms. It is also possible to find anaerobic bacteria because conditions are generated for them to proliferate through the combination of necrotic tissue and low levels of oxygen. For Gentili et al. (2012), the presence of 10 5 or more cells per gram of tissue is a fundamental indicator to confirm that healing will be slow. Goswami et al. (2017) reported more frequent microorganisms to be Staphylococcus aureus, Pseudomonas aeru- mailto:dalia.sanchez@itson.edu.mx Nova Biotechnol Chim (2021) 21(1): e806 2 ginosa, Staphylococcus epidermidis, and Candida albicans. Specifically, S. aureus and P. aeruginosa are the cause of nosocomial infections with an incidence of 20 – 40 % and 5 – 15 %, respectively (Mama et al. 2014). Particularly, P. aeruginosa is an opportunistic gram-negative pathogen capable of causing severe infections in ulcers, in addition to being resistant to multiple antibiotics (Myhrman et al. 2013). Microorganisms of clinical origin develop various mechanisms of resistance to antibiotics due to prolonged therapies. Therefore, the selection of the type and dose of antibiotic is essential to prevent the spread of resistance between microorganisms and promote the healing of ulcers (Cunha et al. 2018). Chitosan is a natural polymer obtained by deacetylation of chitin; it is composed of D- glucosamine and N-acetyl-D-glucosamine (Balti et al. 2017). Its biological applications are due to its biocompatibility, biodegradability, and its nontoxicity (Babushkina et al. 2015; El-Malek et al. 2017). The amine groups of chitosan provide the antimicrobial capacity (Ashrafi et al. 2018) and hemostatic properties. Due to its chemical properties, chitosan has the ability to form films, hydrogels, and fibers (Kiroshka et al. 2014; Khalil et al. 2015). Its potential application in the area of medicine is due to its affinity to aminoglycosides of human tissues (Babushkina et al. 2015). Additionally, it is characterized as being hypoallergenic (Koryagin et al. 2006). The mechanism of action of chitosan is that it alters the cell surface and its permeability, causing the leakage of intracellular substances. This is attributed to the fact that the positive charges of the amino group of chitosan interact with the negative charges of the surface of microorganisms (Demir et al. 2010; Uranga et al. 2019). According to Dragostin et al. (2016), when applying dressings based on chitosan for wound healing, chitosan is degraded into N-acetyl glucosamine, which is used to accelerate the re-epithelialization process. The strategy of using pure chitosan and its topical combinations as an alternative to antibiotics has been proposed to health institutions. In reference to the above, the aim of this work was to evaluate the in vitro effectiveness of films developed with pure chitosan and in mixture with glycerol-honey against gram-negative bacteria isolated from cutaneous ulcers. Honey was incorporated into chitosan films as an adjuvant agent because it has been used to eliminate infections, is a healing agent in skin ulcers (Alam et al. 2014) and is an anti- inflammatory agent (Meo et al. 2017). According to Basualdo et al. (2007), honey promotes the formation of granulation tissue, the growth of the epithelium and healing. In addition, honey has antimicrobial activity against S. aureus, P. aeru- ginosa and K. pneumoniae (El-Malek et al. 2017). Experimental Physicochemical characteristics of chitosan In this research we worked with chitosan obtained in our laboratory (Sánchez-Duarte et al. 2012). The humidity and ash content of the chitosan were 92.42 ± 0.07 % and 0.37 ± 0.02 %, respectively. Its molecular weight was 119.48 kDa and the degree of deacetylation was 84.59 ± 0.87 %. Additionally, the functional groups characteristic of chitosan were identified by Fourier transform infrared spectroscopy (FTIR). Preparation of chitosan membranes The membranes were prepared using the solvent evaporation technique. Six different formulations were developed, three were of pure chitosan at 1, 2 and 3 % in acetic acid at 1 % (w/v), another of 2 % chitosan with 0.2325 ± 0.01 g of glycerin and the other two formulations were 2 % chitosan and honey (95 : 5 v/v), glycerin was added to one of them. The honey was diluted in water (80 : 20 v/v). All the mixtures were homogenized until all the components were fully incorporated. Specifically, 10 mL of each of the formulations were measured independently and a plastic mold was poured and subsequently dried at 40 °C for 24 h. Finally, the membranes were detached from the molds and stored in sterile plastic bags. Sample collection The study was descriptive and cross-sectional. The samples were taken from infected ulcers of patients hospitalized by a doctor specialized in epidemiology. Specifically, the sampling was done Nova Biotechnol Chim (2021) 21(1): e806 3 in the ulcer center with sterile cotton tipped applicator and was introduced in a transport device Stuart (COPAN Transystem R , Brescia, Italy) to keep the isolate in good condition. Once all the samples were collected, they were transported in a hermetically sealed container to a laboratory certified in microbiological analysis and were analyzed in a period no longer than one hour. All patients involved in the trial confirmed their participation with informed consent and the research protocol was approved by the Institutional Ethics Committee. Isolation, identification and antibiotic sensitivity To favor the isolation and identification of microorganisms, samples were seeded by cross- streaking on MacConkey agar (BD Bioxon, Cuautitlán Izcalli, Estado de México, México) for gram-negative bacteria and on Mannitol salt agar (BD Bioxon, Cuautitlán Izcalli, Estado de Mexico, Mexico) for gram-positive bacteria, and the plates were incubated at 37 °C for 24 h. For fungi, the samples were seeded on Biggy agar (BD Bioxon, Cuautitlán Izcalli, Estado de Mexico, Mexico) by sowing and sweeping, and the plates were incubated at 30 °C for 24 h. Subsequently, the identification of microorganisms was performed by the broth microdilution technique. The isolated colonies were taken with a Prompt TM inoculation rod and placed in Prompt TM inoculation bottles until reaching a concentration of 0.08 on the MacFarland standard. The microplates were inoculated with 100 µL in each well and incubated at 37 °C for 24 h. For the gram-negative bacteria, the SIEMENS type 44 microplates were used (B1017-305) and for gram-positive type 33 (B1017-211). Finally, the plates for the identification of the microorganisms were read using LabPro Command Center Software (MicroScan  LabProTM), which indicates the percentage of certainty corresponding to the microorganism identified. Antibiotic sensitivity was measured by the minimum inhibitory concentration (MIC) according to the CLSI (Clinical and Laboratory Standards Institute 2014) criteria. The antibiotics studied were ampicillin, amikacin, ampicillin/sulbactam, cefuroxime, cefotetan, ticarcillin/clavulanate, piperacillin/tazo- bactam, cefazolin, cefotaxime, ceftazidime, imipe- nem, ceftriaxone, moxifloxacin, cefepime, gentami- cin, trimetropin/sulfa-methoxazole, levofloxacin, ciprofloxacin, cefota-xime/clavulanate, ceftazidi- me/clavulanate, tobra-mycin, meropenem and aztreonam. One combination for gram-negative (Klebsiella pneumoniae) is shown, of which the identification was confirmed by biochemical tests and the sensitivity and resistance to antibiotics were evaluated simultaneously. In vitro assays of chitosan membranes against gram-negative bacteria The in vitro tests were determined by the Kirby- Bauer agar diffusion technique. Specifically, for each isolate a dilution in broth was made in an inoculation bottle Prompt TM , and with a sterile swab, the boxes were inoculated in Mueller Hinton agar in seeding by sweeping. Each membrane was divided into disks with a diameter of 16.4 ± 0.10 mm. Three discs were placed with a sterile clamp on the inoculated medium. In addition, two discs of Whatman No. 1 paper impregnated independently with 1 % acetic acid and 0.9 % sodium chloride were placed as blanks. The plates were incubated at 37 ° C for 24 h, and finally, the increase in area and the inhibition halo generated by each membrane was measured. Results Phenotypic identification of microorganisms in skin ulcers In total, 23 ulcers of infected patients were sampled. Gram-negative and gram-positive bacteria were found with 51.61 % and 29.03 % prevalence, respectively. In addition, it was possible to isolate Candida albicans with a prevalence of 19.35 %. Previous studies related to gram-positive bacteria were published, particularly with regard to S. aureus (Escárcega-Galaz et al. 2017). Specifically, this work focused on gram-negative bacteria. From all of the ulcers sampled, 16 gram- negative strains were isolated. Among them, Proteus mirabilis, Escherichia coli, Enterobacter aerogenes, Pseudomona aeruginosa, Klebsiella pneumoniae and Morganella morganii were Nova Biotechnol Chim (2021) 21(1): e806 2 identified. Table 1 shows a comparison of the microorganisms isolated in this investigation with respect to that reported by other authors. Table 1. Comparative analysis of incidences of gram- negative bacteria in skin ulcer. Antibiotic sensitivity of gram-negative bacteria Bacterial resistance to the 23 antibiotics studied is presented in Table 2 (SI-2). Regardless of the phenotype, it was found that all isolated gram- negative bacteria were resistant to ampicillin (CMI ˃16 μg.mL -1 ), ampicillin/sulbactam (CMI ˃16/8 μg.mL -1 cefuroxime (CMI ˃16 μg.mL -1 ), cefazolin (CMI ˃16 μg.mL -1 ), cefotaxime (CMI ˃32 μg.mL - 1 ), moxifloxacin (CMI ˃4 μg.mL -1 ), cefepime (CMI ˃16 μg.mL -1 ) and ciprofloxacin (CMI ˃2 μg.mL -1 ). However, this group of bacteria showed sensitivity to cefotetan (MIC <16 μg.mL -1 ), piperacillin/tazobactam (MIC <8 μg.mL -1 ), imipenem (MIC <4 μg.mL -1 ), cefotaxime/clavulanate (MIC <0.5/4 μg.mL -1 ), ceftazidime/K clavulanate (MIC <0.25/4 μg.mL -1 ) and meropenem (MIC <4 μg.mL -1 ). In vitro assays of chitosan membranes against gram-negative bacteria To determine the potential antimicrobial activity in a clinical setting, chitosan membranes were tested against gram-negative bacteria isolated from ulcers of hospitalized patients. In vitro tests with chitosan-based membranes were adverse for all microorganisms. For all bacterial isolates, the only effect was found by direct contact without the formation of the inhibition halo. Membranes tend to modify their size in relation to the concentration of chitosan. Table 3 shows the percentage of increase in area observed for each bacterium. As the concentration of chitosan in the membrane increased, its percentage increasing in area also increased. Fig. 1 shows an inhibition assay with Escherichia coli in chitosan membranes formulated with glycerol and honey. Fig. 1. In vitro microbiological assay with E. coli in the chitosan 1 % (a), chitosan 2 % + glycerol (b) and chitosan 2 % + honey (c) membranes. Microorganisms This work Mama et al. (2014) Kateel et al. (2018) Malik et al. 2013) Proteus mirabilis 12.50 - - 1.5 Escherichia coli 43.75 20 34.69 27.8 Enterobacter aerogenes 6.25 - - - Pseudomonas aeruginosa 12.50 8 30.61 15.6 Klebsiella pneumoniae 18.75 10 - 5.8 Morganella morganii 6.25 - - 0.7 Klebsiella species - - 12.63 - Proteus species - - 5.10 - Klebsiella oxytoca - - - 7 Proteus vulgaris - - - 3.5 4 Nova Biotechnol Chim (2021) 21(1): e806 3 Table 2. Antibiogram of the 16 isolated skin ulcers. UD- 003 UD- 004 UD- 005 UD- 006 UD- 008 UD- 009 UD- 010 UD- 011 UD- 013 UD- 016 UD- 017 UD- 019 UD- 022 UD- 025 UD- 028 UD- 029 Antibiotic P. mirabilis E. coli E. coli E. coli E. coli E. coli E. aerogenes P. aeruginosa P. mirabilis K. pneumoniae M. morganii K. pneumoniae E. coli K. pneumoniae P. aeruginosa E. coli Ampicillin R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 Amikacin R˃32 R˃32 R˃32 R˃32 R<4 R<4 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 S<4 S<4 R˃32 Ampicillin/Sulbactam R˃16/8 R˃16/8 R˃16/8 R˃16/8 R˃16/8 R˃16/8 R˃16/8 R˃16/8 R˃16/8 R˃16/8 R˃16/8 R˃16/8 R˃16/8 R˃16/8 R˃16/8 R˃16/8 Cefuroxime R˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 Cefotetan R˃32 S<16 R˃32 R˃32 S<16 S<16 R˃32 R˃32 R˃32 R˃32 S<16 S<16 R˃32 S<16 R˃32 R˃32 Ticarcycline/K Clavulanate R˃64 R˃64 R˃64 R˃64 R˃64 R˃64 S<16 R˃64 R˃64 R˃64 R˃64 R˃64 R˃64 S<16 R˃64 R˃64 Piperacillin/Tazobactam R˃64 R˃64 R˃64 R˃64 S<8 S<8 S<8 R˃64 R˃64 S<8 R˃64 S<8 R˃64 S<8 S<8 R˃64 Cefazolin R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 Cefotaxime R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 Ceftazidime R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 S<2 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 Imipenem R˃8 S<4 S<4 S<8 S<4 S<4 S<4 R˃8 R˃8 R˃8 S<4 S<4 S<4 S<4 R˃8 S<4 Ceftriaxone R˃32 S<8 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 R˃32 Moxifloxacin R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 Cefepime R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 Gentamicin R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 S<1 S<1 S<1 R˃8 Trimethoprim/Sulfamethoxazole R˃2/38 R˃2/38 R˃2/38 R˃2/38 R˃2/38 S<2/38 R˃2/38 R˃2/38 R˃2/38 R˃2/38 R˃2/38 R˃2/38 R˃2/38 R˃2/38 R˃2/38 R˃2/38 Levofloxacin R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 S<2 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 R˃4 Ciprofloxacin R˃2 R˃2 R˃2 R˃2 R˃2 R˃2 R˃2 R˃2 R˃2 R˃2 R˃2 R˃2 R˃2 R˃2 R˃2 R˃2 Cefotaxime/K clavulanate R˃4/4 R˃4/4 R˃4/4 R˃4/4 S<0.5/4 S<0.5/4 S<0.5/4 R˃4/4 R˃4/4 R˃4/4 S<0.5/4 S<0.5/4 R˃4/4 S<0.5/4 R˃4/4 R˃4/4 Ceftazidime/K Clavulanate R˃2/2 R˃2/2 R˃2/4 R˃2/4 S<0.25/4 S<0.25/4 S<0.25/4 R˃2/4 R˃2/4 R˃2/4 S<0.25/4 S<0.25/4 R˃2/4 S<0.25/4 R˃2/4 R˃2/4 Tobramycin R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 R˃8 S<1 S<1 R˃8 R˃8 Meropenem S<4 S<4 S<4 S<4 S<4 S<4 S<4 R˃8 R˃8 R˃8 S<4 S<4 S<4 S<4 S<4 R˃8 Aztreonam R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 R ˃16 S<8 R ˃16 5 Nova Biotechnol Chim (2021) 21(1): e806 10 Table 3. Area increase [%] of chitosan membranes. Membrane P. mirabilis E. coli P. aeruginosa K. pneumoniae E. aerogenes M. morganii Ch 1 % 31.9 ± 20.6ª 28.0 ± 21.1ª 30.86 ± 19.3ª 26.1 ± 14.1ª 27.6 ± 5.9ª 32.8 ± 8.8 b Ch 2 % 36.6 ± 24.9 ab 45.4 ± 55.5 bc 36.00 ± 18.4ª 30.7 ± 20.7 ab 46.8 ± 56.2 ab 39.2 ± 4.5 d Ch 3 % 64.6 ± 63.5 c 54.4 ± 27.4 c 39.07 ± 35.7ª 44.8 ± 36.9 ab 58.0 ± 40.6 ab 54.7 ± 19.8 bc Ch 2 % + g 23.2 ± 5.2ª 34.0 ± 17.8 ab 26.10 ± 14.3ª 29.2 ± 14.7 ab 23.6 ± 14.8ª 30.7 ± 5.0 a Ch 2 % + h 32.4 ± 12.1 ab 35.2 ± 12.4 ab 34.52 ± 2.5ª 54.9 ± 52.5 c 30.8 ± 40.9 ab 46.9 ± 0.0 cd Ch 2 % + h + g 51.4 ± 6.3 bc 46.0 ± 14.9 bc 37.99 ± 4.1 a 35.7 ± 38.9 ab 64.9 ± 18.4 c 76.0 ± 18.1 e Initial area: 213.82 mm 2 (n = 6). Discussion Mama et al. (2014) reported that 87.4 % of 150 samples were positive for pathogens, of which 53 % were gram-negative bacteria and 47 % gram-positive. Other authors report that the prevalence for P. aeruginosa is in the range of 20 – 30 % in ulcers (Schmidtchen et al. 2003). Today, a large number of microorganisms have developed resistance to antibiotics, which is why antibacterial drugs are required as new alternatives (Babushkin et al. 2015; El-Malek et al. 2017). Mama et al. (2014) reported that most isolates are resistant to ampicillin and cephalexin with 96 % and 92.4 %, respectively. On the other hand, Malik et al. (2013) reported that resistance to antibiotics among microorganisms is very variable and illustrated it for P. aeruginosa (63.7 %), P. mirabilis (57.5 %), M. morganii (57.5 %), E. faecalis (55.2 %), Acinetobacter sp. (51.9 %), P. vulgaris (50.3 %), E. coli (45.9 %), K. pneumoniae (44.8 %), S. aureus (44.3 %), K. oxytoca (42.9 %), and Coryneform sp. (37.1 %). The antibiotics that they evaluated included penicillins, cephalosporins, monobactams, carba- penems, aminoglycosides, chloramphenicols, quino- lones, and fluoroquinolones, β-lactam inhibitors, macrolides, lincosamides, and glycol-peptide. Finally, Aspiroz et al. (2017) sampled two patients with ulcers in the leg and mentioned that Pseudomonas aeruginosa tends to present a greater recurrence of multi-resistance to antibiotics. The use of antibiotics is only recommended when the patient presents symptoms of infection. Some broad- spectrum medications used for ulcer infection are ampicillin/sulbactam, ticarcillin/clavulanate, coamo- xiclav, clindamycin, quinolone, and cephalosporin. The antimicrobials that have been used to combat the infections are silver sulfadiazine, fusidic acid, metronidazole, sodium chloride, chlorhexidine, and iodine povidone (Howell-Jones et al. 2005). Likewise, when the membranes of chitosan-glycerol are in contact with moisture, they decrease in size due to the membranes being plasticized. Chitosan- honey-bee membranes greatly increase in size by absorbing moisture from the culture medium because honey has the characteristic of being hygroscopic (Sasikala et al. 2013). Through statistical analysis, it was found that there was a significant difference between treatments for each of the microorganisms isolated. While for P. aeruginosa, there was no significant difference between the membranes. Akyzu et al. (2018) reported zones of inhibition of 14.56 ± 0.81 mm for P. microbilis and 14.56 ± 0.92 mm for P. aeruginosa, the zone of inhibition for each microorganism increased by approximately 45 %. On the other hand, Michalska-Sionkowska et al. (2018) performed antimicrobial assays with 1 % chitosan membranes combined with collagen-gentamicin against E. coli, Staphylococcus aureus and Pseudomonas aeruginosa; the zones of inhibition were approximately 25 – 30 mm. As reported by Balti et al. (2017), chitosan does not have the ability to diffuse through agar due to its molecular weight, which is why it does not form halos of inhibition. For chitosan to possess the antimicrobial activity it must be solubilized at a low pH in organic acids (Simunek et al. 2010). In gram- positive bacteria, chitosan has a strong bactericidal 6 Nova Biotechnol Chim (2021) 21(1): e806 3 effect however, in gram-negative bacteria inhibition is considered slower and depends mainly on the molecular weight, degree of deacetylation, concentration and pH of chitosan (Mrázek et al. 2010). The activity of chitosan against gram-negative bacteria is because the molecule tends to become polycationic below its pKa (pH 6.3) (Campana et al. 2017). In addition, the amino group of C2 can interact with the anionic components of negative surfaces, such as lipopolysaccharides and proteins, modifying their cellular permeability. Chitosan at concentrations of 0.1 mg.mL -1 can bind to the negatively charged bacterial surface causing alteration of the cell membrane and leakage of intracellular components. When this polymer is found at high concentrations (2 and 5 mg.mL -1 ), it tends to coat the bacterial surface, limiting the release of intracellular components and mass transfer (Khalil et al. 2015). Bee honey has the potential to fight bacterial infections that have generated resistance to antibiotics and cannot be fought with traditional antibiotics (Kwakman et al. 2011). When the honey is at a pH of 3.1 – 4.5, the glucose is found as gluconolactone, which provides an environment that favors the activity of the fibroblasts and the healing of wounds. Together, the presence of hydrogen peroxide stimulates the generation of fibroblasts and angiogenesis (Alam et al. 2014). In conclusion, the incidence of infected skin ulcers requires safe therapies that promote their healing. One answer to this problem is the use of chitosan membranes. These membranes have confirmed their activity by contact against gram-negative bacteria isolated from ulcers and that are resistant to antibiotics. Therefore, chitosan membranes are a viable alternative as antimicrobial agents for the treatment of infections without the risks involved with antibiotics. Acknowledgment Escárcega-Galaz gratefully acknowledges the CONACYT by Ph.D. scholarship: 417707. This research was funded by the Instituto Tecnologico de Sonora with the project PROFAPI (2021-0011) and by the National Council for Science and Technology (PDCPN2014: 248160). Conflict of interest The authors declare that they have no conflict of interest. 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