Antimicrobial activity of immobilized mycocins in sodium alginate on fecal coliforms Open Access

Mycocins are glycoproteins secreted by some yeasts; these substances have antimicrobial action, causing the death of susceptible microorganisms (Kurtzman et al. 2011; Cappelli et al. 2014; Muccilli & Restuccia 2015).

The production of mycocins can be considered a survival strategy in the environment, which represents an advantage for the producing yeast species among other species in the same habitat (Cappelli et al. 2014), enabling the producing yeasts from being strong competitors and preventing the development of other microorganisms by the release of mycocins in the environment (Stoll et al. 2005). Several species of other yeast genera are reported as mycocinogenic, such as: Saccharomyces, Candida, Cryptococcus, Wyckerhamomyces, Kluyveromyces, and Pichia (Magliani et al. 1997; Pfeiffer et al. 2004; da Silva et al. 2008; Buzdar et al. 2011; Bajaj et al. 2013; Cappelli et al. 2014). Wickerhamomyces anomalus, formerly known as Pichia anomala and Hansenula anomala, is a yeast belonging to the phylum Ascomycota, class Saccharomycetes, order Saccharomycetales, and family Saccharomycetaceae (Kurtzman et al. 2008). It develops easily under different conditions of temperature, osmolarity, and pH and can be isolated from insects, plants, animals, humans, food, soils, and aquatic environments. Some strains of this yeast produce mycocins that inhibit the growth of several microorganisms, such as yeasts, filamentous fungi, bacteria, and parasites (Walker et al. 1995; Fredlund et al. 2002; Walker 2011; Cappelli et al. 2014; Valzano et al. 2016). Due to the broad spectrum of antimicrobial activity and high stability, the W. anomalus mycocins have applicability in processes of the food industry, agricultural biocontrol, and clinical area (Magliani et al. 1997; Passoth et al. 2006; Walker 2011). The antimicrobial activity of mycocins was evaluated through the concentrations of β-glucanases present in the culture of W. anomalus WA40. However, other compounds may also be present in this medium and contribute to the mechanism of action, as reported by Nascimento et al. (2020).

The microbiological quality of water and food can be measured through the presence of bacterial indicators, the coliforms (Noble et al. 2003). These microorganisms are used as reference parameters because their presence indicates potential contamination. Fecal coliforms, also known as thermotolerants, present species of bacteria of the genera: Escherichia, Klebsiella, and Enterobacter (Kagkli et al. 2007).

Escherichia coli is the predominant bacterium in the group of thermotolerant coliforms; its presence in water means fecal contamination. For this reason, it is considered a determining indicator in microbiological quality control tests of water and effluents (Weintraub 2007; WHO 2011). This Gram-negative bacterium, belonging to the Enterobacteriaceae family, is found in the intestinal microbiota, being excreted in human and animal feces. It can be classified as commensal, extraintestinal pathogenic, or enteropathogenic, each associated with various types of infections. It is also responsible for several outbreaks of diarrhea, especially in children (Kaper et al. 2004; Nel et al. 2004; Atidégla et al. 2016). Another point related to the pathogenicity of E. coli is antibiotic resistance. Thus, the discovery of new antimicrobial agents as alternatives to treat and prevent infections by this and other bacteria is of great importance (Chen et al. 2015).

The immobilization of cells and substances demonstrates excellent potential for biotechnological application in the industrial, biocontrol, clinical, and environmental areas, being used in several scientific researches (Khalil & Mansour 2006; King, 2007; Bleve et al. 2011; Hoesli et al. 2011; Jayakumar et al. 2012; Aloui et al. 2015). Cell immobilization techniques help the segregation of a cell in an adverse environment (Westman et al. 2012), ensuring better stress resistance and, consequently, better performance (Nedović et al. 2014).

Problems related to contamination of water sources are much discussed and emphasized. One of the consequences is related to the transmission of several diseases, due to the presence of pathogenic microorganisms in the water. The exacerbated use of antibiotics can cause many strains to become multidrug-resistant to the treatments already used. New antimicrobial alternatives are needed to minimize this. Thus, the present study aimed to assess the antimicrobial activity of free and immobilized mycocines obtained from W. anomalus against various strains of E. coli and fecal coliforms.

For this work, we used a strain of yeast W. anomalus WA40 (access number: KT580792, available at http://www.ncbi.nlm.nih.gov/BLAST). In addition to a standard strain of E. coli ATCC 25922, 45 multi-resistant strains of E. coli isolated from different clinical samples were used (1 tracheal secretion, 1 anal secretion, 1 urethral secretion, 2 abscess secretion, 2 peritoneal secretion, and 38 urine). The assessment of the resistance character was performed using the automated Vitek^® system; the strains showed resistance to more than one of the following antibiotics: Ampicillin, Ampicillin/Sulbactam, Amoxicillin/Clavulanic acid, Cefalotin, Cefuroxime, Cefuroxime, Ceftriaxone, Cefepime, Gentamicin, Nalidixic acid, Ciprofloxacin, Norfloxacin, Nitrofurantoin and Trimethoprim/Sulfamethoxazole.

To obtain the mycocins, we used the W. anomalus WA40 strain, isolated from the soil, belonging to the mycotheque of the mycology laboratory, which was inoculated in modified Sabouraud agar – ASM (2% agar; 1% peptone; 2% glucose; 1.92% citric acid and 3.48% bibasic potassium phosphate – pH 4.7) incubated at 31 °C for 48 h. After this period, a suspension of 10⁶ CFU/mL of the yeast was inoculated into 200 mL of growth broth consisting of 1% peptone, 2% glucose, 1.92% citric acid, and 3.48% bibasic potassium phosphate (pH 4.7). The flasks were incubated at 25 °C for 5 days in a static culture. After this period, the growth broth was centrifuged at 6,000 rpm for 10 min to obtain the broth supernatant with mycocins produced by the yeast; then the supernatant was sterilized by filtration through a 0.22 μm membrane and stored at 4 °C until the in vitro tests were performed.

The determination of the β-glucanases activity of the broth containing the W. anomalus WA40 mycocins was determined as described by Miller (1959), with some adaptations using laminarin 1% (Laminaria digitata), 50 mM acetate buffer, at pH 5.0. A solution containing 62.5 μL of the supernatant was prepared with W. anomalus WA40 and 125 μL of laminarin 1% and incubated at 37 °C for 10 min. We withdrew 100 μL from the solution and added 100 μL of 3,5-dinitrosalicylic acid to stop the reaction. The solutions were incubated in boiling water for 5 min, then 500 μL of sterile distilled water was added, and the reading of the reaction product (reduced sugar) was at 550 nm. For the blank, the same test solution was used without laminarin. An enzyme unit (U) was defined as the amount of protein required to produce 1 μmol of reducing sugar per minute (U/min/mL). The test was performed in triplicate.

The quantification of proteins present in the supernatant containing the mycocins was analyzed by the Bradford (1976) method using Bovine Albumin as the standard curve, and the equation of the line was used to calculate the total concentration of proteins in mg/mL. The specific activity of β-glucanases was calculated by dividing the concentration of enzymatic activity by the concentration of proteins.

The antimicrobial activity of the mycocins was according to the concentrations of β-glucanases present in the supernatant of the WA40 culture of W. anomalus. A total of 45 strains of multidrug-resistant E. coli and ATCC25922 E. coli were used. The susceptibility of the 46 bacterial strains was evaluated using the microdilution in the broth method described by the Clinical and Laboratory Standards Institute – M7-A10 (CLSI 2015) with some modifications. Suspensions of each strain of E. coli were prepared in Mueller-Hinton (MH) broth and adjusted to obtain a final concentration of 1–9 × 10³ CFU/mL. The concentrations of β-glucanases used were 0.1, 0.2, 0.4, and 0.8 U/mg prepared in sterile water. Sterile microplates of 96 flat-bottomed wells were used. For each well of the microplate column, 100 μL of the bacterial suspensions were applied. In each line of the microwell plate, 100 μL of the respective concentrations of β-glucanases were added. In one of the microplate lines, growth control was performed, represented by the suspension of 100 μL MH broth with the distinct bacterial strain and 100 μL of sterile modified Sabouraud broth (CSM). Another line represented sterility control containing only 200 μL modified sterile Sabouraud broth. The plates were incubated at 35 °C, and after 48 h, their readings were performed, observing the presence or absence of visible growth as a reference for growth control. A 10 μL aliquot of the wells in which there was no turbidity was inoculated into nutrient agar to confirm the absence of growth. The test was performed in triplicate.

We used different concentrations of sodium alginate: 1.0, 1.5, 2.0, 2.5 and 3.0% (w/v), and calcium chloride at 0.05, 0.1, 0.2, 0.3 and 0.4 mol/L for immobilization of the mycocins.

For each concentration of sodium alginate, flasks containing 12.5 mL of supernatant containing the mycocins (0.8 U/mg of β-glucanases) and 12.5 mL of previously sterilized sodium alginate solution were prepared. The resulting mixture was kept under stirring for 15 min at 60 rpm and then dripped in flasks containing 100 mL of CaCl₂ solution at different concentrations. They were maintained under stirring of 60 rpm for the development of granules with the immobilized mycocins. After dripping the entire solution of mycocins and sodium alginate in calcium chloride, the granules resulting from immobilization were kept under stirring for another 5 min and at rest during the same time. The immobilized mycocins were washed in sterile water three times for complete removal of the calcium chloride solution. After immobilization, the resulting granules were evaluated for their weight, size, and physical appearance.

The activity of immobilized mycocins was assessed against E. coli bacteria ATCC25922. A 1–5 × 10⁶ CFU/mL saline suspension of E. coli was prepared, and aliquots of 10 μL were added to flasks with 50 mL of a solution containing 1% peptone and 1% glucose. In one of the flasks, 50 mL of granules were added with immobilized mycocins, and the other flask without the granules was used as a positive control for bacterial growth. The flasks were kept static at 35 °C. Then an aliquot of 1 μL of each flask was inoculated into nutrient agar plates, and incubated at 35 °C for 24 h, after the CFU/mL count was performed.

For this experiment, the mycocins were immobilized using 2% sodium alginate and 0.2 mol/L calcium chloride. The total volume of granules resulting from the immobilization of mycocins under the conditions above was 50 mL, and they were packed in bags made of nylon yarn. Five water samples contaminated with feces were used in their respective bacterial concentrations: 100, 240, 250, 300, and 350 CFU/mL. The bags were added to flasks containing water contaminated with feces, incubated statically at 35 °C. For each sample, a flask containing only samples of water contaminated with feces was used as a positive control of bacterial growth and incubated under the same conditions. An aliquot of 1 μL was removed from each flask and inoculated into MacConkey agar plates incubated at 35 °C for 24 h. After this period, the CFU/mL of the different colonies present in each plate were counted and compared with the growth of the samples from the control flasks.

The toxicity test was performed using the technique of Meyer et al. (1982) with some adaptations. A. salina eggs were incubated in sterile seawater at 28 ± 2 °C for 48 h under continuous aeration and illumination. After hatching, 10 nauplii (larvae) were housed in tubes containing 1,000, 100 and 10 ppm of supernatant in sufficient quantity for 5 mL of seawater. The maximum toxicity control consisted of NaOH 1 mol L⁻¹, and the nontoxic control included only seawater. The tubes were incubated at 28 ± 2 °C for 24 h, and the test was performed in triplicate. The count was performed with the aid of an optical microscope, and the motility of the larvae was considered, being classified as alive or dead.

The granules resulting from the immobilization of the mycocins with the different concentrations of sodium alginate and calcium chloride had an average weight of 0.075 g and an average diameter of 4.5 mm. The appearance of the granules was also assessed, and the granules of immobilization using the concentrations of 2.0, 2.5, and 3.0% sodium alginate presented better results, forming granules of spherical shape, uniform, and with a smooth surface.

Mycocins immobilized at concentrations of 2% sodium alginate and 0.2 mol/L calcium chloride showed better inhibitory activity on the E. coli strain ATCC25922. After 24 h of incubation, the total CFU/mL of the E. coli strain ATCC25922 decreased over the days when compared to the positive control of bacterial growth. After 8 days of testing, bacterial growth was not verified in this treatment. The concentrations of calcium chloride at 0.3 and 0.4 mol/L also showed inhibitory activity, but the action of mycocins started after 48 h of incubation. Mycocins that were immobilized using calcium chloride at concentrations of 0.05 and 0.1 mol/L showed later inhibitory activity.

A. salina is a marine microcrustacean used in toxicity tests related to plant extracts (Arcanjo et al. 2012). The Artemia test was performed in this study because it is easy to manage, fast, has a low cost, and is related to the potential of not using animals in toxicological trials (Rajabi et al. 2015).

When performing the tests, the mycocins revealed no toxicity in the microcrustaceans tested up to the concentration of 1,000 ppm, values that are standardized for the plant test. Thus, the low toxicity of the mycocins stands out. However, no studies with mycocins were found in the literature for this type of research.

Antibiotic resistance is a global problem, and the wide variety of resistant infectious agents is a growing threat to public health due to the rapid spread of multi-resistant bacteria through water, such as E. coli (WHO 2015).

In the present study, all multidrug-resistant strains of E. coli were sensitive to the action of W. anomalus WA40 mycocins; this effect was also found on the standard strain of E. coli ATCC 25922, used in broth microdilution tests in different proportions (Figure 1).

Olstorpe et al. (2012) used intact H. anomala cells against various enterobacterial species, and over the days of treatment, found a reduction in the number of CFU of E. coli strains in the presence of yeast.

Paris et al. (2016) used mycocins produced by W. anomalus WA40 and verified their action against Candida albicans strains. The authors observed that all strains tested were inhibited. The mycocins are more active against other yeasts but present antagonistic behavior to other microorganisms, such as bacteria (Comitini et al. 2005; Meneghin et al. 2010), and can thus be used as an alternative antimicrobial agent. The antibacterial activity of mycocins has been demonstrated against both Gram-negative and Gram-positive bacteria. Junges et al. (2020) reported that mycocins from W. anomalus completely inhibited multidrug-resistant isolates of Acinetobacter baumannii from human samples. Calazans et al. (2021) observed inhibition of Staphylococcus aureus isolates from meat by mycocins. Additionally, Nascimento et al. (2022) demonstrated the inhibition of carbapenemase-producing Klebsiella pneumoniae by mycocins. Further comparisons can be drawn from other studies that highlight the broad-spectrum activity of mycocins. Al-Qaysi et al. (2017) demonstrated that mycocins produced by Debaryomyces hansenii DSMZ70238 exhibited potent antimicrobial effects against both Gram-positive and Gram-negative bacteria, including E. coli and S. aureus. Similarly, Giovati et al. (2021) reviewed the medical applications of Wickerhamomyces yeast killer toxins, emphasizing their potential as therapeutic agents due to their effectiveness against clinically relevant bacterial pathogens, including multidrug-resistant strains.

Results of the inhibitory action of mycocins on E. coli strains were obtained by Chen et al. (2015), who used the mycocins of two yeast strains, Saccharomyces cerevisiae, and Kluyveromyces marxianus.

Considering that the studies of yeast-secreted antimicrobial compounds are still in the development phase, exploring and elucidating the activity of such compounds that are produced by these microorganisms is, in fact, a strategy to find new candidates for antibiotics (Hatoum et al. 2012; Svahn et al. 2012).

According to Covizzi et al. (2007), microbial immobilization offers many advantages, such as a high concentration of immobilization, better resistance to contamination, stimulation of the production and secretion of secondary metabolites and the physical and chemical protection of bioactive cells and compounds.

Alginate is one of the most used polymers for the immobilization of cells and enzymes due to its characteristics: easy handling, nontoxic properties, biocompatibility, availability, and low cost (Gombotz 1998; Kawaguti & Sato 2008; Westman et al. 2012; Duarte et al. 2013). Thus, immobilization using this polymer does not affect the performance and activity of the immobilized.

The mycocins immobilized in this study showed antimicrobial activity against E. coli and bacteria present in water contaminated with feces. For Elizei et al. (2014), the immobilization method using sodium alginate does not affect the biological processes of microbial cells, since it is a nontoxic polymer. According to Kawaguti & Sato (2008), the activity of immobilized cells will depend on the surface size of the spheres, the porosity of the gel formed, and the hydrophilic characteristics of the support.

Sodium hypochlorite is widely used in water purification due to its effectiveness and low cost. However, it has cytotoxic effects on tissues (Hsieh et al. 2020; Böhle et al. 2022) and poses significant environmental risks, including adverse effects on aquatic fauna, and the formation of byproducts like trihalomethanes, which are carcinogenic (Heidari 2019; Sriboonnak et al. 2021; Sun et al. 2023). In contrast, mycocins have demonstrated no hemolytic effect when in contact with human erythrocytes (Paris et al. 2016), and in the present study, mycocins showed no toxicity in A. salina, suggesting minimal environmental impact. Given that sodium alginate is low cost and the immobilization technique is simple and scalable, mycocins immobilized in sodium alginate present a promising and environmentally safer alternative for wastewater treatment.

For Goh et al. (2012) and Gombotz (1998), the factor that determines the average diameter of the spheres is the size of the extrusion nozzle, concentration, and viscosity of the sodium alginate used. The granules resulting from the immobilization of the mycocins presented variations concerning the physical aspect according to the different concentrations of sodium alginate used, a result not observed when varying the concentrations of calcium chloride, which influenced the beginning of the inhibitory activity of immobilized mycocins. Won et al. (2005) reported that the different concentrations of calcium chloride did not affect the immobilization; only the variation of the concentration of sodium alginate did. In this work, different concentrations of sodium alginate and calcium chloride were used, and the results obtained are in agreement with other scientific research, so when varying the concentrations of sodium alginate and calcium chloride, there were differences in the physical aspect and activity of immobilized material (Becerra et al. 2001; Quiroga et al. 2011; Andriani et al. 2012; Rehman et al. 2013).

Waterborne disease transmission is the leading cause of death in many countries, and there is an urgent need for the assessment of disinfection methods considering the need for new approaches to process improvement (Li et al. 2008). According to the World Health Organization, new alternatives to treat water and effluents in order to remove the microbial load indicator of fecal pollution are being researched since the effects that these microorganisms can cause on the population are considered of great importance, taking into account, mainly, the spread of diseases through water, thus directly affecting public health (WHO 2011).

The yeasts that produce mycocins have characteristics that enable their application in various biotechnological processes linked to food, rations, biopreservation, fermentation, and wastewater treatment (Marquina et al. 2002; Walker 2011; Schneider et al. 2012).

The yeast W. anomalus develops easily in different conditions of temperature, osmolarity, and pH; thus, such characteristics turn it into a candidate for bioremediation in contaminated waters (Fredlund et al. 2002; Walker 2011). Immobilization techniques using microalgae and bacteria have been studied in scientific research as an alternative to the treatment of contaminated water and sewage (Covarrubias et al. 2012; Cruz et al. 2013).

In this work, W. anomalus WA40 mycocins immobilized using sodium alginate as a polymer inhibited the development of bacteria present in water contaminated with feces. Aloui et al. (2015) found that whole cells of W. anomalus immobilized in biofilms of sodium alginate showed total antimicrobial activity on the growth of Penicillium digitatum. Therefore, we can suggest that immobilization does not affect the action of this yeast, either by immobilizing the yeast or the mycocins it produces.

Considering the alginate immobilization technique and the action of W. anomalus mycocins, the results obtained in this work show that the mycocins obtained from W. anomalus WA40 presented antimicrobial activity on several strains of E. coli and fecal coliforms, even being immobilized; thus, the immobilization of mycocins would be a possible alternative for the treatment of water contaminated with fecal material.

Future studies should assess the efficacy of immobilized mycocins not only against other thermotolerant coliforms but also against pathogenic microorganisms. The prolonged antimicrobial activity of immobilized mycocins and their rapid microbial elimination make them suitable for various wastewater treatment systems, including those from pig and dairy cattle farming and even hospital wastewater.

We concluded with this work that the mycocins produced by the W. anomalus WA40 strain were able to inhibit multi-resistant strains of E. coli in addition to the standard E. coli ATCC 25922 strain. By varying the concentration of sodium alginate, there was a change in the physical appearance of the granules resulting from the immobilization of mycocins, a result not observed when varying the concentrations of calcium chloride. The immobilized mycocins showed inhibitory activity compared to the standard E. coli strain ATCC 25922 when immobilized at concentrations of 2% sodium alginate and 0.2 mol/L calcium chloride; the inhibitory activity started after 24 h of incubation. Under these immobilization conditions, immobilized mycocins also showed antibacterial action against coliforms present in the samples of water contaminated with feces; in this experiment, all samples did not show growth of bacteria after 48 h of testing. Mycocins obtained from W. anomalus WA40 were able to inhibit E. coli and bacteria present in water contaminated with fecal material, either by using these substances in free or immobilized form.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.