A Brazilian Bentonite Previously Treated with Benzalkonium Chloride Reduces Pseudomonas Aeruginosa Growth

Research Article

J Bacteriol Mycol. 2017; 4(2): 1050.

A Brazilian Bentonite Previously Treated with Benzalkonium Chloride Reduces Pseudomonas Aeruginosa Growth

Nones J¹*, Savi GD², Müller L¹, Trentin AG³, Angioletto E², Soares C¹, Riella HG¹ and Nones J³

¹Department of Chemical Engineering, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil

²Post-Graduate Program in Materials Science and Engineering, UNESC, Criciúma, Santa Catarina, Brazil

³Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil

*Corresponding author: Jader Nones, Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil

Received: April 19, 2017; Accepted: May 05, 2017; Published: May 12, 2017


Pseudomonas aeruginosa can pose a serious threat to public health, as it causes a wide variety of clinical syndromes, including sepsis, pneumonia, meningitis, conjunctivitis and skin infections. Since early times, various types of bentonites have been used in the traditional medicine of most countries to avoid inflammatory reactions caused by different bacteria. Therefore, the primary objective of this work was to study the effects of natural bentonite and bentonite previously treated with an organic salt (benzalkonium chloride - BAC302) on the in vitro growth of Pseudomonas aeruginosa. For this purpose, after the organophilic treatment, we have previously characterized these bentonites (natural and BAC302) using Scanning Electron Microscopy (SEM) and the procedures by Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH). After characterization, the bentonite samples were submitted to the agar dilution method and the disc diffusion test to evaluate the effect on the growth of Pseudomonas aeruginosa. Our results show no differences in the surface morphology of the two materials tested (natural Brazilian bentonite and BAC302). However, the organic treatment reduces the specific surface area and increases pore diameter of BAC302. The growth of Pseudomonas aeruginosa was found to be reduced after treatment with BAC302, but not after treatment with natural bentonite. Another aim of the present work was to observe the effects of natural bentonite and BAC302 on fibroblast cultures. The two materials were not found to be toxic to fibroblast cells when dosed at the concentrations of 0.3-0.9 mg/ mL. This study contributes to a better understanding of the effects of bentonite on mammalian tissue, and the results may contribute to development of novel treatment strategies for P. aeruginosa infections.

Keywords: Bentonite; Benzalkonium chloride; Fibroblasts; Pseudomonas aeruginosa


Pseudomonas aeruginosa is a nonfermentative, gram-negative bacterium responsible for a wide variety of diseases, including sepsis, pneumonia, meningitis, conjunctivitis, respiratory infections, dermatitis and soft tissue infections [1,2,3]. Moreover, it has been associated with both environmental reservoirs and healthcare workers’ carriage [4,5].

Despite the availability of antibiotics, P. aeruginosa infections continue to represent a public health threat. In the USA, the Centers for Disease Control and Prevention have reported 1.7 million hospital acquired infections yearly caused by all types of gram-positive (G+) and gram-negative (G-) bacteria, including Pseudomonas, resulting in 99,000 deaths every year [6].

As an aggravating factor, Pseudomonas aeruginosa is a notoriously - resistant bacterium that is increasingly refractory to antimicrobial chemotherapy [7,8,9]. Also, resistance to multiple classes of antimicrobials (multidrug resistance) increases in this organism. This can be caused by chromosomal mutations and/ or the acquisition of resistance genes via horizontal gene transfer [8,10,11]. In order to control this bacterium, alternative, safe and cost effective treatments should be developed. Compounds prepared with clay, such as bentonites, are attracting great attention in the field of biology and medicine because of the synergies found between its biopharmaceutical and technological features [12,13,14]. Bentonites have been shown to protect mammal cells from several insults [15,16,17]. Kevadiya et al. [18], for example, showed that clay minerals reduce genotoxic effects and facilitate drug delivery. In addition, various types of clay minerals have been used in the traditional medicine of most countries in order to treat a variety of diseases, mainly related to skin problems [19,20,21].

Alkylammonium cations, including Benzalkonium Chloride (BAC), is one of the most important quaternary ammonium compounds used for disinfection of surfaces in medical care applications as well as in the food and glue industries, because of their antibacterial activity [2,22,23,24]. Traditional pharmaceutical nasal sprays and drops require preservatives that prevent microbial contamination, and BAC is by far one of the most used preservatives in aqueous formulations [25,26] to prevent different infections.

We have recently demonstrated that natural bentonite and bentonite previously treated with benzalkonium chloride (BAC302) increase aflatoxin B1 adsorption significantly compared with untreated groups [17]. However, knowledge on the effects of bentonites associated with surfactants (BAC302) on cells or cell cultures is scarce [17,27]. Moreover, to our knowledge, there are no data reported about in vitro studies on the effects of organo-modified bentonite (BAC) on Pseudomonas aeruginosa bacteria.

This paper is the first report to describe the Pseudomonas aeruginosa bactericide effects of a Brazilian bentonite previously treated with organic salt (BAC302). Furthermore, it reports the results from toxicity studies on organic bentonites (BAC302) using fibroblast cells as a mammal culture model in order to test the biosafety of the product created, when applied to cells that form the epithelial tissue.

Materials and Methods

Reagents and chemicals

Dulbecco’s Modified Eagle Medium (DMEM-F12), penicillin, and streptomycin were purchased from Invitrogen. Fetal Bovine Serum (FBS) and trypan blue solution were purchased from Lonza (Verviers, Belgium). Dimethyl sulfoxide (DMSO), 4’,6-diamidino- 2-phenylindole dihydrochloride (DAPI) and benzalkonium chloride were purchased from Sigma–Aldrich (St. Louis, MO, USA). The materials for antimicrobial assays culture medium Mueller Hinton (MH) agar, Tryptic Soy Broth (TSB) and Brain Heart Infusion (BHI) were supplied by Himedia Laboratories (Bhaveshwar Plaza, Mumbai, India). Pseudomonas aeruginosa (ATCC 27853) was purchased from Newprov Products for Laboratory (Pinhais, Paraná, Brazil). All other chemicals were purchased from commercial sources and were of analytical grade. Bentonite samples, characterized by Nones et al. [28,29], were collected in the city of Criciúma, located in the State of Santa Catarina, Brazil. The bentonite clays (natural or treated) were kept in a stock solution diluted in DMSO at -20°C.

Preparation of organobentonites

Organobentonite (BAC302) was synthesized by Nones et al. [17] according to the following process: 5g of bentonite was first added to 50mL of distilled water at 30°C and stirred until they were thoroughly dispersed. Desired amounts of benzalkonium chloride were mixed in 50mL distilled water at 30°C for 30min. Then the modifying agents were added to the bentonite suspension under vigorous stirring. The mixed suspensions were stirred at 30°C for 4h and then stored at room temperature (around 25°C) overnight. After that, the resulting products were washed with distilled water and dried at 80° C. The added amounts of BAC were 2% of the bentonite’s weight.

Bentonite characterization

Surface morphology of bentonites was determined by scanning electron microscope (SEM) JEOLJSM- 390LV [17]. The textural properties of bentonites were determined by Brunauer-Emmett– Teller (BET) using automated Quanta chrome Instruments (Autosorb-1). For this purpose, the samples were out-gassed at 250°C for 12h under nitrogen prior to adsorption measurement with the multi-point method. Pore distributions and pore volume were calculated using the adsorption branch of the N2 isotherms based on the Barrett–Joyner–Halenda (BJH) pore size analyzer.

Antimicrobial activity

Disc diffusion method: The modified disc diffusion method was performed according to the recommendation of the methodology of the Clinical and Laboratory Standards Institute (CLSI), approved standards M02-A10 [30]. The inoculum for the agar dilution method was prepared with growing Pseudomonas aeruginosa (ATCC 27853) to the turbidity standards. Therefore, five well-isolated colonies of the same morphology type were selected from an agar Mueller Hinton (MH) plate culture grown by 24 to 48h and transfer into a tube containing 5mL of tryptic soy broth. The culture was incubated at 35°C by 4h and adjusted until it reached the turbidity standard measured by absorbance at 625nm from 0.08 to 0.13 (1 x 108 CFU/ mL) for Pseudomonas aeruginosa, equivalent to a 0.5 MacFarland standard. Within 15 min after adjusting the turbidity of the inoculum suspension, a sterile cotton swab was dipped into the suspension and spread on the entire sterile agar MH surface. The medium surface was allowed to dry and the natural bentonite and BAC302 was deposited on top of the dry surface.

All plates were incubated at 37°C for 24h. After incubation, the presence zone of growth inhibition around the antimicrobial materials samples was observed and its diameter in millimeters was measured.

Agar dilution method: The agar dilution method was performed according to the recommendation of CLSI, approved standards M07-A8 [31]. The natural bentonite and BAC302 (in powder form) were incorporated into 25 mL of MH agar medium at 45° C and poured in Petri dishes in triplicates to achieve different concentrations (0.3, 0.6, 0.9, 1.2 and 1.5 mg/mL). The medium was mixed thoroughly with the materials and allowed to solidify at room temperature. A growth control containing only medium without the materials was performed and another growth control containing the maximum percentage of BAC incorporated in natural bentonite was performed (data not shown).

The inoculum standards were prepared similarly to the method mentioned above to obtain the concentration of 108 CFU/mL for Pseudomonas aeruginosa. The adjusted culture was diluted in 1:10 in sterile saline to obtain a concentration of 107 CFU/mL and after that 2μL drop was applied to the agar surface (~104 CFU per spot). Finally, the inoculated plates were maintained at room temperature until the moisture in the inoculum spots had been absorbed, inverted and then incubated at 35 ± 2° C for 16 to 20h. The Minimum Inhibition Concentration (MIC) was recorded as the lowest concentration of the antimicrobial material that completely inhibits bacterial growth.

Macrodilution broth method: The macrodilution broth method was performed according to the recommendation of CLSI, approved standards M07-A8 [31]. The natural bentonite and BAC302 (in powder form) were incorporated into tubes containing 2.7mL of Brain Heart Infusion (BHI) medium broth to achieve different concentrations (0.3, 0.6, 0.9, 1.2 and 1.5 mg/mL). A growth control containing only medium broth without the materials was performed. Indeed, another growth control containing the maximum percentage of BAC incorporated in natural bentonite was also realized (data not shown). The inoculum standards were prepared similarly to the method mentioned above to obtain a bacterial suspension with 108 CFU/mL concentrations. The adjusted culture was diluted in sterile broth and added in the tube containing BHI at the concentration of 105 CFU/mL. The macrodilution tubes were incubated at 35° C ± 2° C for 16 to 20h under stirring. Thereafter, an aliquot (10μL) was taken and applied on the MH agar medium and then incubated at 35 ± 2°C for 16 to 20h. Finally, the number of Colony-Forming Units (CFUs) was determined and the Minimum Bactericidal Concentration (MBC) was defined as the minimal concentrations of compounds required to kill the organisms.

Fibroblast cell cultures

Mouse embryonic fibroblast (3T3) was obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle medium (DMEM-F12) supplemented with 10% of fetal bovine serum, 100 units/mL penicillin, and 100mg/mL streptomycin. 3T3 cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and routinely kept in a logarithmic growth phase through trypsinization twice a week. In addition, the culture media were changed every 2 days. For the assays, the cells were plated in 96 well plates at a seeding density of 30 thousands cells per cm2. Cell cultures (3T3) were incubated with DMSO/Benzalkonium chloride (control group), 0.3-1.5 mg/mL of natural bentonite or BAC302. After 48 h of treatment, cell viability by trypan blue or cell count analysis were determined by DAPI staining. The final concentration of DMSO in the cell culture was a maximum of 1%.

Trypan blue assay

The viability of fibroblast (3T3) cells were determined by trypan blue as described by Youn et al. [32] and Nones et al. [17,28,29]. After exposure, the cells were collected by trysination, and stained with 0.4% trypan blue solution at room temperature for 3min. The cells were then counted using a hemocytometer and a light microscope. At least one thousand cells were found and the percentages of unstained (viable) and stained (nonviable) cells were determined.

Cell count analyses

Morphology and viability of 3T3 fibroblasts were also analyzed by DAPI nuclear binding dye and optical and fluorescence microscopy (Olympus IX71). The cells were harvested and fixed with 4% paraformaldehyde for 15min without temperature control. After washing in PBS, cell nuclei were stained with DAPI and visualized and counted. At least ten fields were measured per well.

Quantitative and statistical analysis

Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test or two-way analysis of variance (ANOVA) followed by Bonferroni post-tests, using the GraphPad Prism 5.0 software. P < 0.05 was considered statistically significant. The experiments were performed in triplicate and each result represents the mean of at least three independent experiments.


Morphology and chemical composition does not change after treatment with BAC

By using Scanning Electron Microscopy (SEM), we found that benzalkonium chloride did not change bentonite morphology (Figure 1A, 1B). Both samples (natural and BAC302) show individual particles, most of which had clearly recognizable contours that tended to form thick and large agglomerates. These data are in agreement with our previously results, whereby we could not see differences in natural or organic bentonites using SEM [17].