Microbicide Action of IAA and IAA/HRP on Staphylococcus Aureus

Action of indole-3-acetic acid (IAA) in absence and presence of horseradish peroxidase (HRP) was evaluated on the virulence factors produced by Staphylococcus aureus: biofilm producer; enterotoxins producer (seb-gene, SEB); methicillin resistance (mecA) and control-stain by: i) dose-response on microorganism growth and minimum inhibitory concentration (MIC) by spectrophotometry at 625nm; ii) membrane damage by flow cytometry using fluorescence detector at 610nm and 530nm; iii) biofilm production by spectrophotometry at 570 nm; iii) antioxidant enzyme activity as catalase and superoxide dismutase by spectrophotometry at respective 240nm and 550nm. Dose-response on microorganism growth was seen and MIC by IAA alone was 40mM for biofilm, 50mM for SEB and control-stain, and 60mM for mecA; HRP potentiated the IAA action on biofilm, SEB and mecA. Subinhibitory IAA concentration increased membrane damage for only biofilm and control-strain; however in presence of HRP the injury was potentiate in all stains. Except for biofilm, all strains showed similar increase in catalase activity by IAA and IAA/HRP. Superoxide dismutase activity was similarly increased in presence of IAA and IAA/HRP for all stains. IAA alone or combined with HRP presents microbicide action on S. aureus with different pathological characteristics; probably involving reactive oxygen species since increased activity of antioxidant enzymes.


INTRODUCTION
Staphylococcus aureus is a pathogenic microorganism capable of causing a range of life-threatening and mild diseases, including septicaemia, meningitis, toxic shock syndrome, food poisoning and skin abscess [1].The virulence factors produced by S. aureus include biofilm production, enterotoxin production and resistance to antibiotics such as methicillin.The production of biofilm may be responsible for the persistence of bacterial infections, by increasing adherence of the microorganism and providing protection against antimicrobial substances [2].S. aureus produces a variety of extracellular protein toxins including enterotoxins related to food poisoning [3].Various types of staphylococcal enterotoxin (SE) are produced by S. aureus; types A (SEA), B (SEB), C (SEC) and D (SED) are commonly implicated in staphylococcal food poisoning [3].The protein SEB, a potent toxin which has also gained the status of a biological warfare agent, is expressed by the seb gene of S. aureus [3].Virulence-related resistance to methicillin in Staphylococcus spp. is commonly conferred by the mecA gene that encodes a cell wall protein with low affinity for this class of antimicrobial [4,5].Studies have identified new compounds for use in possible alternative therapeutic strategies for pathogen combat.
Diverse mechanisms have been reported for cytotoxicity from the oxidation of IAA by HRP enzyme, including the involvement of indolyl, skatolyl and peroxyl radicals and reactive oxygen species (ROS) such as superoxide  − ) and hydrogen peroxide (H2O2) [11,12].Reactive species act on microbial targets causing multiple concurrent disruptive events, thereby preventing development of resistance, and allowing inactivation of a wide spectrum of microorganisms, independent of their profiles of resistance to classical antimicrobial agents [8,12].
The aim of this study was to evaluate the action of IAA and IAA/HRP on S. aureus strains with different characteristics conferred by biofilm production, enterotoxins production by seb-gene presence (food poisoning) and methicillin resistance by mecA gene presence, and a strain negative for these virulence characteristics.

Materials and Reagents
All reagents and enzymes were of analytical grade and were obtained from Sigma Chemical Company (St. Louis, MO, USA).Culture medium was obtained from Oxoid (Basingstoke, Hampshire, England).

IAA and HRP Preparation
A stock solution of IAA (100 mM; 17.5 mg/mL) was prepared in tryptic soy broth (TSB) and NaOH (5 M) was added for complete solubility of this acid, and thereafter the pH value was adjusted to 7.4 by using HCl (25%, v/v) [13].HRP solution (1 µM) was prepared in Phosphate Buffer Saline (PBS) and the pH value was adjusted to 7.4, from a stock solution of HRP (1000 µM) [10].

Dose-response of IAA on S. Aureus Growth
Microorganism growth was available by optic density (OD) value at 625 nm and by a qualitative technique using resazurin assay [27] with some modifications.To both methods the microorganism (3.0 x 10 3 CFU/mL) was placed in polystyrene plate using TSB and 0.5, 4, 10, 20, 30, 40, 50, 60 and 70 mM of IAA in absence or presence of HRP 1 µM.Negative control incubations without the microorganism were performed.For OD assay, after incubation of microorganism at 37 º C under agitation for 18 hours, microplates OD was determinate using a microplate reader at 625 nm (Beckman Coulter DU-800 (UV/Vis).The results were expressed as OD values versus IAA concentration.Inhibition percentage of microbial growth was determinate to each IAA concentration in relation with microorganism in absence of IAA.For resazurin assay, a resazurin solution (6.76 mg/mL) was placed in each well and the plate incubated at 37°C under agitation for 18 hours.The change of purple (oxidized state of resazurin) to pink color (reduced state of resorufin) is recorded as positive (growth of bacteria).

Biofilm Formation
The biofilm production was assessed by the OD value from adhering bacteria and stained with violet crystal evaluated at 570 nm using a microplate reader (Multiskan FC -Thermo Scientific) [28].The S. aureus biofilmproducer (ATCC 6538) and control stain (ATCC 29213) were used.The microorganism (3.0 x 10 3 CFU/mL) was placed in polystyrene plate using TSB supplemented with 0.25% glucose in presence of 30 mM (subinhitory) and 40 mM (MIC) of IAA in absence or presence of HRP 1 µM.Negative control incubations without the microorganism were performed.Bacterial suspension was incubated for 18 hours at 37 o C. So, each well was aspirated and was carefully washed three times with PBS and dried at 60 minutes in inverted position.Next, the wells were stained with 0.5% violet crystal solution for one minute.For qualitative assay the plate was photographed and analyzed.The intensity of blue color in polystyrene plate wells indicated bacterial adhesion and has been grouped into three Eurasian J Anal Chem 3 / 9 categories according to the color: no adherent (OD ≤ 0.120), weakly bonded (OD 0.120 to 0.240) and strongly adherent (OD ≥ 0.240).

Membrane Damage
For membrane integrity a sub inhibitory IAA concentration was used.The IAA concentration was 30 mM to stain biofilm and SEB and 40 mM to mecA and control stains.Cell membrane integrity was analyzed by FACSAria flow cytometry equipment (BD Biosciences, USA).This instrument is equipped with an argon ion laser for excitation of the fluorescent dyes, providing 15 mW at 488 nm.Filter set up was standard.In this assay, the suspension of S. aureus (3.0 x 10 4 CFU/mL) was incubated with propidium iodide (PI) (43 µM) and thiazole orange (TO) (420 nM) for 5 minutes at room temperature according to the BD Cell Viability Kit.PI red fluorescence and TO green fluorescence emitted were collected using respective band-pass filters at 610 nm and 530 nm.The data were analyzed using the BD FACSDiva Software (BD Biosciences, USA).To determine differential staining of live (green fluorescence) and dead microorganism (red fluorescence)) in the presence of TO and PI, similar assays were carried out also through a mixture of live microorganism and microorganism that was heat killed by exposure to 70 o C for 10 min [29].

Antioxidant Enzyme Activities
Antioxidant enzyme activities were evaluated in S. aureus incubated with a sub inhibitory IAA concentration, where 30 mM IAA for biofilm and SEB stains and 40 mM IAA for mecA and negative control stains were used.For enzymes extraction, the microorganism was incubated with lysostafin (20 IU/mL) for 30 minutes at 37ºC with constant agitation and was centrifuged at 13,000 x g for 15 minutes.Next, the supernatant was kept in the ice to quantify the protein content and enzyme activity using a spectrophotometer (DU-800, Beckman).The catalase activity was measured at 240 nm, following H2O2 reduction [30].The SOD activity was determined based on its inhibitory effect on the rate of superoxide-dependent reduction of nitro blue tetrazolium by xanthine-xanthine oxidase at 550 nm [31].The protein content was determined by the described method for [32], using bovine serum albumin for construction of the curve.

Expression of Results and Statistical Analysis
Data are expressed as means ± standard deviation.Comparisons between groups were initially performed by analysis of variance (ANOVA).The alpha level (significance level related to the probability of rejecting a true hypothesis) was set at 0.05.Significant differences were then compared using Tukey's test with a significance coefficient of 0.05.

Dose Response of IAA on S. Aureus Growth
Inhibition of bacterial growth related to IAA concentration in the absence and presence of HRP was observed (Figure 1).All S. aureus strains cultured in the presence of IAA (Figure 1A) or IAA/HRP (Figure 1B) showed normal growth below 10 mM in both cases, evaluated by optical density.However, concentrations up to 20 mM of IAA and IAA/HRP inhibited microorganism growth for all strains except for mecA which was sensitive even at low IAA concentrations.
The growth inhibition percentage of S. aureus cultivated in the presence of IAA and IAA/HRP was determined; the values are shown in Table 1.Inhibition of microbial growth by IAA in the absence or presence of HRP was evidenced firstly for mecA (10 mM), followed by both biofilm and SEB (20 mM) and finally the control strain (30 mM).An MIC condition, when IAA concentration inhibited 90% of bacterial growth, was determined.Alone, IAA showed an MIC of 40 mM for the biofilm strain, 50 mM for both SEB and control strains and 60 mM for the mecA strain.HRP enzyme potentiated the inhibitory effect of IAA on microorganism growth; IAA MIC of 30 mM for biofilm, 40 mM for SEB and 50 mM for mecA strain were seen.However, for the control strain, MIC was 50 mM IAA for IAA alone or in the presence of HRP.

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The results show that the S. aureus biofilm-producing strain was most sensitive to exposure to IAA or IAA/HRP, showing 100% inhibition of microorganism growth in the presence of IAA at lower concentrations than for the other strains.The mecA strain was more resistant to IAA exposure than the others, 60 mM IAA being necessary to achieve 94% inhibition of microbial growth, although it presented sensitivity, showing a reduction in microbial growth from 10 mM IAA.
In MIC conditions, the number of viable bacteria was reduced, so for subsequent assays IAA concentrations below the MIC (sub inhibitory concentrations) were used.Thus, in the present study, the sub inhibitory IAA concentration was 30 mM for biofilm and SEB strains, and 40 mM for mecA and control strains; in these conditions, around 50% to 60% bacterial growth inhibition was found.
The resazurin assay showed a purple colour in the culture medium for microorganisms grown in the presence of IAA and IAA/HRP which inhibited microbial growth; a similar colour was seen in the presence of gentamicin (9.4 μg/mL).Culture medium for microorganisms cultured in the absence of IAA showed a pink colour, indicating

IAA Action on S. Aureus Biofilm-producing Strain
Bacterial growth of the S. aureus biofilm-producing strain was more sensitive to IAA exposure; however, biofilm formation by this strain was not affected by IAA.Biofilm production was assessed by the bright blue colour resulting from the interaction between the crystal violet dye and microorganisms adhered to the culture plate.In the present study, a similar colour was observed for the S. aureus biofilm-producing strain in the absence and presence of IAA or IAA/HRP (data not shown); this parameter was quantified as an OD of 0.34 ± 0.03 for microorganism alone and as 0.44 ± 0.03 and 0.33 ± 0.03 for microorganism exposed to IAA (30 mM) in the absence and presence of HRP, respectively.In contrast, the control strain showed OD values of 0.03 ± 0.01, 0.03 ± 0.01 and 0.03 ± 0.01, respectively, for the strain incubated alone and in the presence of IAA or IAA/HRP.Values are expressed as mean and standard deviation for six different experiments (n = 6).

IAA Action on Membrane Integrity and Antioxidant Enzyme Activity
S. aureus exposed to IAA and IAA/HRP had reduced membrane integrity (Table 2).IAA alone increased damage 1.7 and 2.5 times for the biofilm and control strains, respectively, compared to microorganism alone.However, IAA/HRP treatment increased membrane damage for all strains compared to microorganism alone.Membrane damage was increased 4.0, 3.4, 14.8 and 4.4 times compared to microorganism alone for biofilm, SEB, mecA and control strains, respectively.The presence of HRP potentiated the action of IAA on membrane damage 1.8 times for biofilm, 1.6 times for SEB and 3.6 times for mecA strains.For the control strain, no significant difference was found between IAA and IAA/HRP.An increase in antioxidant enzyme activity was seen for microorganism in presence of IAA and IAA/HRP (Table 3).IAA alone showed a significant increase of CAT activity, 1.3, 5.2 and 1.9 times for SEB, mecA and control strains, respectively, compared to microorganism alone.The presence of HRP did not alter the effect of IAA on CAT activity for SEB, mecA and control strains.For the biofilm strain, no alteration in CAT activity was seen for IAA or IAA/HRP.Microorganism alone, incubated in the absence of IAA or IAA/HRP, showed CAT activity for all strains; however, the S. aureus biofilm-producing strain had greater CAT activity than the other strains.SOD activity increased 1.6, 0.8, 0.9 and 0.7 times for biofilm, SEB, mecA and control strains, respectively, incubated in the presence of IAA alone compared to microorganism alone.However, similar values were found for microorganism incubated in the presence of IAA/HRP.All strains showed SOD activity in the absence of IAA or IAA/HRP.

DISCUSSION
Antimicrobial agents from natural compounds have been studied as alternative therapies due to increasing microbial resistance to current therapies.Cunha et al. (2015) observed that reduction of P. zopfii viability was related to IAA concentration.An in vitro study showed antimicrobial activity of auxin on S. aureus and other microorganisms [6].
Similar to IAA molecules, indole-3-carbinol and 3-methylene-2-oxindole have a toxic action on prokaryotic and eukaryotic cells [10,33].Indole-3-carbinol has antimicrobial activity in vitro by disrupting the structure that makes up the cell membrane in various species of microorganism such as the Gram-positive bacteria S. aureus, S. epidermidis and Enterococcus faecium; Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa; and fungi such as Saccharomyces cerevisiae, Trichosporon beigelii and Candida albicans [34].
In the present study, the results show that IAA alone or combined with HRP reduces S. aureus growth for strains with different virulence characteristics; a subinhibitory IAA concentration reduced membrane integrity and increased antioxidant enzyme activity (CAT and SOD), but no DNA fragmentation was seen.These results corroborate those of other studies in which a drastic reduction of P. zopfii growth by 1 mM IAA associated with 1 µM HRP was observed without, however, changing DNA integrity [9].Similar results were found by Pugine et al. (2010) for S. aureus from subclinical bovine mastitis exposed to the same concentrations of IAA and HRP.Others studies show IAA as antifungal on Aspergilus fumigates, Candida albicans and Rhizopusoryzae [35].
Antioxidant enzymes such as CAT and SOD have been shown to be sensitive indicators of increased oxidative stress, and increased activity of these antioxidant enzymes is known to serve as a protective response to eliminate ROS [27,36].In the present study, the inhibition of S. aureus growth by exposure to IAA or IAA/HRP may be due to prooxidant action.In fact, the IAA/HRP system has been explained by several authors as leading to an increase in formation of ROS which present themselves as potent cytotoxic radicals [11,26,33].Studies show that reactive cytotoxic species can cause irreversible damage to molecular microorganism constituents or even destroy them [37].Furthermore, exogenous microbial metabolites have been shown to reduce CAT activity in S. aureus, and the authors suggest that CAT activity depletion could be related to the mechanism of cell death [38,39].Moderate oxidative stress conditions increase the antioxidant activity of the enzymes; however, in extreme oxidizing conditions, enzymatic activity is impaired [13].In the present study, an increase in antioxidant enzyme activity was observed in S. aureus exposed to exogenous substances.
For the biofilm-producing strain, CAT activity was not changed by the presence of IAA or IAA/HRP, which seems to be related to the high basal activity of CAT in the S. aureus biofilm-producing strain, suggesting that oxidative stress produced by these exogenous substances does not result in increased CAT activity.Even so, the deleterious effects of IAA and IAA/HRP on the S. aureus biofilm-producing strain were detected by changes in bacterial growth and by membrane damage.
OD values greater than 0.240 classify the biofilm-producing microorganism as strongly adherent, and OD values smaller than 0.120 classify it as nonadherent [28].Our results show that the strong adherence of the S. aureus biofilm-producing strain was not affected by exposure to IAA or IAA/HRP (respective OD of 0.34 and 0.44).The physiological characteristics of biofilm organisms and biofilm structure can confer inherent resistance to antimicrobial agents [40].However, in the present study, IAA alone or combined with HRP induced membrane damage and inhibited bacterial growth of the S. aureus biofilm-producing strain without affecting biofilm formation.
The spectrophotometric technique employed in the present study to estimate the bacteria number related to OD (625 nm) value is widely used in microbiology studies [3][4][5][6][7][8][9][10]18].Recently, OD (570 nm) value produced by a dye Eurasian J Anal Chem 7 / 9 incorporated into bacteria has been used to determine biofilm formation by same microorganisms [14,15,28].In present study, OD determination was shown to be efficient to evaluate the action of IAA and IAA/HRP on the S. aureus growth; as well as these compounds action on biofilm formation.

CONCLUSION
Our studies show that IAA alone or combined with HRP has microbicide action on S. aureus with different virulence factors; probably involving reactive oxygen species by increasing of antioxidant enzymes activities.The membrane damage of S. aureus shows a possibility for growth inhibition this microorganism in presence of IAA.These findings can contribute to the understanding of bacterial cell death, which may be useful for the discovery of new bactericidal and bacteriostatic products.

Figure 1 .
Figure 1.Dose response evaluated by IAA in absence and presence HRP on growth inhibition of S. aureus biofilm producer (biofilm), seb-gene presence (SEB), mecA gene presence (mecA) and control-stain (control = no biofilm produce and absence of seb-gene and mecA gene).Results as mean and standard error (n = 6) Relative percentage of membrane damage calculated in relation to total microorganism (above 1000 cells).Different lowercase letters represent significant difference in the same row (P≤0.05).The values are presented as the mean and standard deviation from six experiments (n=6).IAA = 30 mM to biofilm and SEB stains; IAA = 40 mM to mecA and negative stains.HRP = 1 µM for all tests Vaz et al. / Antimicrobial Action of indole-3-acetic acid 6 / 9

Table 1 .
Relative percentage of inhibition growth of S. aureus biofilm producer (biofilm), seb gene presence (SEB), mecA gene presence (mecA) and control stain (control = no biofilm produce and absence of seb gene and mecA gene) evaluated in different

Table 2 .
Relative percentage of membrane damage from S. aureus biofilm producer (biofilm), seb-gene presence (SEB), mecA presence (mecA) and control stain (no biofilm produce and absence of seb gene and mecA gene).(-) absence of IAA or HRP and (+) presence of IAA or HRP

Table 3 .
Enzyme activity of catalase (CAT) and superoxide dismutase (SOD) from S. aureus biofilm producer (biofilm), seb gene presence (SEB), mecA presence (mecA) and control stain (no biofilm produce and absence of seb gene and mecA gene) evaluated in absence of IAA (-), presence of IAA (+) and presence of HRP (+)Different letters in the same row represent significant difference (P≤0.05) for each enzyme.The values are presented as the mean and standard error from nine experiments (n=9).CAT is expressed as mmol/min per mg of protein and SOD is expressed as U/mg of protein.IAA = 30 mM to biofilm and SEB, IAA = 40 mM to mecA and control stain.HRP = 1 µM for all tests