Acetic Acid Increased the Inactivation of Multi-drug Resistant Non-typhoidal Salmonella by Large-Scaffold Antibiotic

Abstract

Salmonella is a gram-negative bacterium with intrinsic resistance to large-scaffold antibiotics due to the presence of an outer membrane. Based on the mode of action of the organic acids in outer membrane disintegration, and consequently, an enhancement in cell permeability, a combination of acetic acid and a large-scaffold antibiotic is it evaluated. Therefore, the aim of this study is to assess the combination of different levels of acetic acid with vancomycin, in order to determine whether or not the organic acid may overcome the cell wall and the intrinsic resistance in multi-drug resistant Salmonella. Screening of five wild-type Salmonella strains and one clinical strain was performed to select the strain more resistance to acid inhibition. Acetic acid was tested at 2.0, 1.75, 1.50, and 1.25% levels, separated or combined with 8 µg/mL vancomycin dose. An aliquot was collected after exposure and inoculated into the brain and heart infusion agar. The plates were counted and the data analyzed by ANOVA and a posthoc Tukey test (p < 0.05). The results indicate that 1.25 and 1.50% levels did not affect the vancomycin inactivation of multi-drug resistant Salmonella. However, at levels of 1.75 and 2.0%, an increase in microbial reduction is observed. Also, 2% level acetic acid and vancomycin had a threefold increase compared to vancomycin alone. Therefore, the use of acetic acid as prior treatment for Salmonella increased the inactivation rate of vancomycin. The combination of organic acid and antibiotics is a potential tool to overcome cases of antimicrobial resistance.

Introduction

Salmonella is a gram-negative bacterium that has been classified in more than 2600 serovars with different phenotypic profiles [1, 2]. Although Salmonella non-typhoidal infection is mainly related to gastrointestinal symptoms in humans, the bacteria have also been isolated from skin lesions [3] and several cases of meningitis [4]. An important trait of gram-negative species is the presence of an outer membrane that protects the cell against harsh extra-cellular milieu [5]. In addition, outer membranes have a β-barrel responsible for maintaining cellular homeostasis and selecting the molecules that enter the cell [5]. This fact is important because it represents a hurdle to large-scaffold molecules, such as many antibiotics, in penetrating the bacteria [6, 7].

An alternative for overcoming this challenge is to cause damage in the cell envelope using substances with a capacity to permeate the membrane. As described by Alakomi et al. [8], permeabilizer substances allow other compounds to penetrate the cell. Consequently, they have an increased susceptibility to hydrophobic antibiotics, detergents, lysozyme, or bacteriocins. In this way, Alakomi et al. [8] suggested that organic acids are potent outer membrane disintegrating agents, with the capacity to cause lipopolysaccharides (LPS) release and consequently more permeability in the cell.

Based on the mode of action and chemical characteristics of organic acids, we hypothesized that Salmonella exposition to acetic acid might enhance the permeability of its plasmatic membrane. Thus, favoring the influx of antimicrobial agents such as vancomycin, into the bacterial cell and increasing the efficacy of vancomycin in inactivating resistant strains, since that resistance does not mean immunity to the substance. Therefore, the aim of this study is to combine different levels of acetic acid with vancomycin for overcoming the intrinsic resistance of Salmonella to large-scaffold antibiotics, allowing the development of a novel approach to inactivate MDR Salmonella.

Materials and Methods

Screening Using Disk-Diffusion Test for Selection of MDR Salmonella

Screening is performed in order to select a MDR Salmonella strain with higher resistance to acetic acid (Merck^® Darmstadt, Germany). Five MDR strains previously isolated from chicken meat by Cunha-Neto et al. [9], and one Salmonella ATCC strain (ATCC 6539), were tested by a disk diffusion test [10]. To achieve this goal, the disk diffusion assay for acetic acid was performed in a five-step analysis. First, each strain was placed on Müller–Hinton broth 2 (MH; Himedia^®, Mumbai, India) and incubated between 2 and 4 h, up to 0.5 MacFarland scale (approximately 10⁵ CFU/g). Posteriorly, the strains were streaked onto Müller–Hinton 2 agar (MH; Himedia^®, Mumbai, India), and 10 µL acetic acid disks of diffusion (LB; Laborclin^®, São Paulo, Brasil) were placed on the agar surface. The plates were then incubated for 24 h at 37° C. Finally, the halos caused by the acetic acid were measured, and the statistical comparison was applied to the data.

Minimum Inhibitory Concentration of Acetic Acid

MIC was performed by a microdilution test on a 96-well plate. Briefly, 0.2 mL of Brain Heart Infusion (BHI), with the Salmonella strain at 10⁵ CFU/g, was included in each well. The following acetic acid levels were tested: 25, 20, 15, 10, 5, 4, 3, 2, and 1% (100% = 602.9 g/L), with 15-min exposure time. Subsequent, serial dilutions with 0.1 mL were inoculated into BHI agar (Kasvi^® São José dos Pinhais, Brazil). After an incubation period of 24 h at 37° C, plates were counted. In the present study, we selected the highest level, allowing maximum level growth to be tested. The three sequential, lower levels were also tested. The pH of the selected concentrations of acetic acid ranged from 3.1 at the highest dosage (25%) to 4.6 at the lowest one (1%). This pH is directly associated with the pKa constant for acetic acid [11].

Determination of the Mean Dose of Vancomycin Inhibition

To determine the vancomycin antibiotic level to be tested (Sigma-Aldrich^® St. Louis, United States), we performed a trial using a concentration of 8 μg/mL for 4 h of exposure, following the recommendations of CLSI [12]. Vancomycin was previously diluted in a Tryptone Soya Broth medium (Sigma-Aldrich^® St. Louis, United States). Briefly, an initial Salmonella count was performed by serial dilutions with 0.1 mL of BHI broth inoculated in BHI agar surface. After that, 1 mL of Salmonella cultured in BHI broth was exposed for 4 h to 1 mL of vancomycin at 8 µg/mL. Posteriorly, plate counting was performed by serial dilutions and inoculating 0.1 mL of the medium into Petri dishes containing BHI agar. The concentration was determined with the objective of obtaining a minimum reduction, with a statistical difference in the control group (without acid and without antibiotic).

Interaction Between Acetic Acid and Vancomycin in the Inhibition of MDR Salmonella

The interaction tests were performed with four levels of acetic acid: 2.0%, 1.75%, 1.5%, 1.25%, and 8 μg/mL of the antibiotic vancomycin. Moreover, control treatment was included with only vancomycin (without acetic acid). The activation of the bacterium was performed as previously described. For the standardization of the initial count in all treatments, we used a tube containing 50 mL of inoculated BHI of Salmonella to perform the experiments. In addition, an initial count of Salmonella was performed with 0.1 mL of BHI broth without treatment by serial dilutions and was inoculated into BHI agar. The acetic acid was placed in the tubes according to the determined dosage and remained for 15 min at room temperature (approximately 25 °C). Subsequently, 0.1 mL aliquot was plated by serial dilutions on BHI agar to determine the reduction by acetic acid, and another 1 mL aliquot was removed and included in the TSB medium with vancomycin (pH 7.1) and incubated at room temperature (approximately 25 °C). An aliquot was plated on BHI agar, and the counts were determined. Differences between the initial and the final counts of Salmonella were calculated, consequently inactivation results.

Statistical Analysis

Data of acetic acid and vancomycin combination were initially tested for normality of standardized residuals and homogeneity of variances. Once these assumptions were met, data were analyzed by one-way ANOVA (disk-diffusion) and two-way ANOVA (acetic acid and an antibiotic). In case of significant differences, means were separated by Tukey test (p < 0.05).

Results

Screening Test, Determination of Acetic Acid Levels and Vancomycin Dose

The result obtained in the screening test was described in Table 1. Salmonella O:4,5 strain showed a significant difference (p < 0.05) to Salmonella ATCC strain. However, no significant difference was detected between wild-type strains. Nevertheless, in the study performed by Cunha-Neto et al. [9], Salmonella O:4,5 strain also showed the higher antimicrobial resistance profile, with three pharmacological classes of resistance. Therefore, this strain was selected for the subsequent analysis.

Table 1.

Inhibition of Salmonella non-tripoidal strains by acetic acid (1.02 mg/µL) in disk-diffusion test

Strain	Mean of halo inhibition (mm)1
Salmonella (O:4,5)	16.0a ± 1.0
S. Agona	17.7ab ± 3.2
S. Abony	18.0ab ± 3.0
S. Infantis	18.7ab ± 3.2
S. Shwarzengrund	19.0ab ± 3.1
S. Typhimurium (ATCC 23564)	22.7b ± 2.5

Lowercase letter indicates statistical difference between strains. The (±) represents standard error

¹All the results were analyzed by the Tukey test at the 5% level (p < 0.05)

With relation to the acetic acid level, the minimum inhibitory concentration obtained was 3%. However, in the present study, we use the higher concentration of the acid, in which there is microbial growth, as the maximum level to be combined with the antibiotic vancomycin. Thus, the concentration of 2.0% was used along with the other 1.75%, 1.50%, 1.25%, and 0%. After determining the acetic acid levels, the dosage of vancomycin to be tested was determined as the one responsible for reducing approximately 1.4 log CFU/mL in the MDR Salmonella, i.e., 8 μg/mL.

Interaction Between Acetic Acid and Vancomycin

The results of the interaction between acetic acid and vancomycin are presented in Fig. 1. When we analyze the treatments with acetic acid only, it is possible to observe that in all levels of acetic acid, there is a statistical effect (p < 0.05), when compared to the control group (without acetic acid and vancomycin). This result reinforces the capacity of acetic acid at penetrating the cell of gram-negative bacteria. However, the concentration of acetic acid has an influence on antimicrobial effects; this fact is achieved in the final count of cells that did not show a significant difference at 1.25 and 1.50% levels. Thus, the significative increase in the inactivation cells is observed in 1.75 and also in 2.0% levels (p < 0.05).

Fig. 1.

Interaction between acetic acid and vancomycin on the final concentration of multi-drug resistant Salmonella. Capital letters indicate statistical differences between acetic acid concentrations within each level of vancomycin (with and without). Lowercase letters indicate statistical differences between vancomycin levels (with and without) within each concentration of acetic acid. Adjusted means (LS means) were separated by Tukey adjusted method (p < 0.05)

Regarding the interaction of acetic acid combined with vancomycin, the results indicate that 1.25 and 1.50% levels with vancomycin (8 μg/ml) did not differ significantly when compared to the control group with vancomycin only (Fig. 1). However, in the levels of 1.75 and 2%, there was an increase in microbial reduction with a significant difference (p < 0.05) to 1.25 and 1.50% levels. Moreover, at 2% acetic acid and vancomycin, there was a threefold increase compared to control treatment with vancomycin alone. Furthermore, about treatments with and without vancomycin, it is possible to verify that vancomycin at 1.75 and 2.0% levels of acetic acid there was an additive effect in the reduction of Salmonella MDR strain.

Discussion

Results obtained in the present study are in agreement with the mechanism described by Alakomi et al. [8], where organic acid to permeabilize the outer membrane in bacterium cell, and the mechanism of action of acetic acid is illustrated in Fig. 2. Moreover, the increase in cell permeability using organic acids is also described by Zhitnitsky et al. [13], in which a synergistic capacity is verified when acetic acid is combined with transition metals. This result corroborates the hypothesis of increased cell influx and, consequently, an increase in the capacity of bacterial inhibition. Our results suggest that in the fraction containing 1.75 and 2.0% of acetic acid, there was an increase in cellular permeability, and the combination of vancomycin resulted in the highest percentage of inhibition (Fig. 1).

Fig. 2.

Mechanism of action of organic acid in outer membrane to permeabilize the in-bacterium cell. (a) Acetic acid penetrates into the cell and dissociates, resulting in a concentration of ion H⁻ (with antimicrobial effect), to restore the intracellular pH; the cell pumped the ion out. However, this mechanism causes a disturbance of the cell and leads to an energetically unfavorable process [21]; (b) Another mechanism of organic acid is liberation of lipopolysaccharide from the outer membrane [8]; (c) The permeability action caused by organic acid in the outer membrane, to allow that lipophilic substance has access to the inside of the cell [8]; (d) Acetic acid promotes permeability in the cell, and vancomycin acts in blocks of peptidoglycan polymerization by binding to the peptidyl-d-alanyl-d-alanine termini of peptidoglycan precursors [22]. Moreover, vancomycin also inhibits the synthesis of ribonucleic acid, with action in gram-negative strains [19]

However, despite the combined effect of acetic acid and vancomycin, the important point is that, in levels of 1.25% and 1.50% of acetic acid, the reduction in the counts of Salmonella was similar in the groups with and without vancomycin. We hypothesize that in sub-lethal levels or with a low inhibitory effect, acetic acid can function as a stage of cell stress and, consequently, increase the expression of regulatory and resistance genes. In a study carried out by Yuk and Marshall [14], it was found that exposures to sub-lethal levels of organic acids were able to induce simulated gastric fluid resistance, which corresponds to a mode of cell inactivation.

Our results indicate that the above 1.75% level of acetic acid, in combination with an antibiotic, is an alternative to antimicrobial resistance problems, which currently represents a public health crisis [15]. Currently, the antimicrobial resistance in Salmonella represents a direct risk to public health, having cases of outbreaks reported in several countries [16, 17]. In the present study, we used an isolated strain of chicken meat with a multidrug-resistant characteristic (resistance to three pharmacological classes), representing a direct risk to the consumer [9]. In addition, this strain represents a potential for gene resistant dispersion among other strains of Salmonella spp.

Moreover, resistance to vancomycin is reported in a study performed by Thung et al. [18], in which all Salmonella isolates were found to be resistant to vancomycin. This resistance shows an alarming trend to public health authorities, because vancomycin is a narrow-spectrum antibiotic, being a glycopeptide mainly active in gram-positive strains with cell wall inhibition, but also inhibits the synthesis of ribonucleic acid with inhibition in gram-negative strains [19]. In addition, vancomycin is known as the antibiotic of last resort, used when other drugs have not been effective [20]. Resistance to this drug alerts to the presence of strains that may not be controlled by antibiotics, and it is necessary to develop novel combinations for cellular inactivation of these isolates.

However, a challenge in the use of acetic acid in in vitro assay is due to its higher ability to dissociate at a more alkaline pH; therefore, it is necessary that the use of the acid occurs under controlled pH, with total dissociation of acetic acid occurring at a pH between 5.0 and 5.5 [11]. Thus, we used the exposure for 15 min to simulate the action of organic acid and the time to total dissociation in the human and animal intestine and skin. Moreover, in our study, the maximum pH used is within this range (maximum 4.6), so the acid could penetrate the cell and dissociate in contact with the cytoplasm. Further studies using other organic acids as propionic, butyric, and sorbic acids, and the simultaneous use of the acids with the antibiotic, should be performed. Novel strategies can be performed to combine the acid with the antibiotic, such as the development of drugs with topical action and oral use. However, other studies evaluating in vivo, time of exposure, and the collateral effects should be performed. Nevertheless, the result of the present study indicates that the combination may be a potential tool to overcome cases of antimicrobial resistance.

The use of acetic acid as prior treatment for Salmonella increases bacterial inactivation by vancomycin. This increase was more evident when acetic acid was used in concentrations higher than 1.75%. At the level of 2%, acetic acid treatment promoted an inactivation rate three times higher than that obtained exclusively with the antibiotic. However, at levels below 1.25%, the organic acid did not improve antibiotic efficacy and may have functioned as a cellular stress factor that induced pre-adaptation responses.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.