Biogenic silver nanoparticles: in vitro activity against Staphylococcus aureus methicillin-resistant (MRSA) and multidrug-resistant coagulase-negative Staphylococcus (CoNS)

Abstract

Multidrug-resistant (MDR) bacteria are one problem in health since the therapeutic alternative are reduced. For this, the application of nanotechnology through functionalized nanoparticles, like a biogenic silver nanoparticle (Bio-AgNP), obtained by biological synthesis, emerges as a possible alternative against the MDR bacteria. This study aimed to evaluate the antibacterial and antibiofilm activity of Bio-AgNP obtained for biological synthesis by Fusarium oxysporum strain 551 against methicillin-resistant Staphylococcus aureus (MRSA) and MDR coagulase-negative Staphylococcus (CoNS) isolates. Bio-AgNP has activity against S. aureus ATCC 25904, Staphylococcus epidermidis ATCC 35984, and MDR isolates, with minimal inhibitory concentration (MIC) ranging from 3.75 to 15 μg.mL^-1 and minimal bactericidal concentration (MBC) from 7.5 to 30 μg.mL^-1. In the membrane leakage assay, it was observed that all concentrations tested led to proteins release from the cellular content dose-dependently, where the highest concentrations led to higher protein in the supernatant. The 2×MIC of Bio-AgNP killed ATCC 35984 after 6h of treatment, and ATCC 25904 and S. aureus (SA3) strains after 24h of treatment. The 4×MIC was bactericidal in 6h of treatment for all strains in the study. The biofilm of MDR isolates was inhibited in 80.94 to 100% and eradicated in 60 to 94%. The confocal laser scanning microscopy (CLSM) analysis demonstrated similar results to the antibiofilm assays. The Bio-AgNP has antibacterial and antibiofilm activity and can be a promising therapeutic alternative against MDR bacteria.

Introduction

Multidrug-resistant (MDR) strains of Staphylococcus spp., such as methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative Staphylococcus (CoNS), are widely distributed in hospitals and communities, which can lead to infections that are difficult to treat, with limited therapeutic resource [1, 2]. This resistance profile is mainly attributed to the expression of the mecA gene, being responsible for resistance to β-lactam drugs, such as penicillin, cephalosporins, monobactams, and carbapenems, in addition to other drugs [3]. These bacteria can form biofilms on biotic surfaces, making treatment difficult, and increasing the rates of morbidity, mortality, and clinical complications [4–6].

Considering the problem of infections involving these bacteria, the research, and development of new drugs for their control is considered a high priority [4]. In this sense, several studies have been performed applying nanotechnology from functionalized nanoparticles (NPs) as a possible alternative [5]. The NPs allow the delivery of a bioactive compound to the site of interest with reduced side effects. In addition, the surface of these compounds plays an important role, allowing their dispersion and colloidal stability in physiological media, biocompatibility, and also the presence of reactive groups and biocides, such as silver (Ag) [6]. These metals have a broad spectrum of action and can be a viable alternative against different microorganisms, even MDR [7].

The NPs can be synthesized using different methods, but their production using ecological methods, such as biological synthesis, is of great interest because they are safer, simpler, and scalable [8–10]. Among them, biogenic silver NPs (Bio-AgNP) have different biological properties, such as antioxidant [11], anti-inflammatory [12], anticancer [10], larvicide [13], antimicrobial [14–16], and antibiofilm [17].

The Bio-AgNP has a broad spectrum of action, low toxicity, and can be used in combination with conventional antimicrobials, being a viable alternative in the control of microorganisms, including the MDR [7, 18]. Thus, the objective of this study was to evaluate the antibacterial and antibiofilm activity of Bio-AgNP synthesized from the fungus Fusarium oxysporum strain 551, against MRSA and MDR CoNS isolates.

Material and methods

Synthesis of biogenic silver nanoparticles (Bio-AgNP)

The Bio-AgNP used in our study was obtained by the biological method of fungus-mediated synthesis [19]. The method consisted of the reduction of silver nitrate by Fusarium oxysporum strain 551 at the Molecular Genetics Laboratory of ESALQ-USP (Piracicaba, São Paulo, Brazil). The production method of Bio-AgNPs was patented (Patent, 2006, PI 0605681–4A2). The fungus was grown in 90 mm Petri dishes in a medium containing 0.5% yeast extract (HiMedia®, Mumbai, India), 3% malt extract (Acumedia®, Michigan, USA), and 1.5% agar (Kasvi®, Italy) at 28°C for 7 days. After growth, the fungal biomass was separated from the culture medium with a pipette and added in sterile distilled water, and 0.1 g of mycelium for every 1 mL water was added and incubated at 28°C on an orbital shaker (150 rpm) for 72 h. Subsequently, the components were fractionated by filtration and centrifugation at 1860 × g for 5 min. The supernatant was mixed with 10 mM AgNO₃ (Sigma-Aldrich®, St. Louis, USA) and incubated at 28°C for 10 days. The aliquots of Bio-AgNPs were measured by absorbance at 440 nm in a UV-Vis spectrophotometer (Varian Cary 50 Probe). The diameter and zeta potential (mV) of Bio-AgNPs were determined by photon correlation spectroscopy using ZetaSizer Nano ZS; the zeta potential of the Bio-AgNPs used in this study was −32.9, and the size was 100 nm.

Bacteria strains

Ten S. aureus (SA) and MDR coagulase-negative Staphylococcus (CoNS) clinical isolates belong to the Laboratory of Bacteriology and Bioassays (LaBBio), Federal University of Pelotas (UFPel, Pelotas, RS, Brazil) and were kindly provided by the Microbiology Laboratory of School Hospital, Federal University of Pelotas (UFPel, Pelotas, RS, Brazil) (biorepository accession numbers: SA3, SA4, SA5, SA6, SA9, CoNS3, CoNS5, CoNS6, CoNS16, and CoNS17) which were used in this study. These strains were identified by VITEK® 2 GP System (bioMérieux, France). The reference strains used in this study were obtained from American Type Culture Collection (ATCC) provided by Oswaldo Cruz Foundation (FIOCRUZ, Rio de Janeiro, Brazil), which are Staphylococcus aureus ATCC 25904 and Staphylococcus epidermidis ATCC 35984.

Antibiotic susceptibility

The antibiotic susceptibility of isolates to cefoxitin, gentamicin, and oxacillin (Laborclin®, Brazil) was evaluated by the disk diffusion (DD) method, and for vancomycin (VAN) was determinated by minimal inhibitory concentration (MIC) [20]. The bacteria strains were grown on Brain Heart Infusion Agar (BHI - Kasvi®, Italy) at 37°C for 18 h, and a bacterial inoculum suspension was prepared in 0.9% NaCl solution (1×10⁸ CFU.mL^-1). For the DD, this suspension was spread on the Mueller–Hinton Agar (Sigma-Aldrich®, St. Louis, USA), and after drying, the antimicrobial disks were added to the plates and incubated at 37°C for 16–18 h. The results were made by measuring the inhibition halo in mm and compared to the parameters of CLSI [20].

Identification of mecA gene

The isolates were confirmed as MDR by amplifying the mecA gene using the Polymerase Chain Reaction (PCR) technique. Primers mecA-F (CAT CCT ATG ATA GCT TGG TC) and mecA-R (CTA AAT CAT AGC CAT GAC CG) were used to amplify the region corresponding to mecA gene (342 pb). For reaction, the following reagents were used: 13 μL Go Taq® Colorless Master Mix (Promega, EUA), 1 μL of each primer, 5 μL genomic DNA obtained by frozen colonies [21], completing the final volume to 25 μL with ultrapure water. The amplifying conditions were 94°C for 4 min; 30 cycles: 94°C for 30s, 47°C for 30 s, 72°C for 1 min; 72°C for 4 min. PCR products were visualized using 1% agarose gel electrophoresis [22].

Minimal inhibitory and bactericidal concentration (MIC and MBC)

The bacterial isolates were submitted to different concentrations of Bio-AgNP (0.029 to 30 μg.mL^-1) and VAN (0.5 to 64 μg.mL^-1), which was performed by CLSI [20] in 96-well polystyrene microtiter plates. For this, 50 μL of the bacterial inoculum (1×10⁸ CFU.mL^-1) was added to a 50 μL of BHI broth (Kasvi®, Italy) with different concentrations of Bio-AgNP and VAN. As a control was used only the culture medium (negative control), culture medium plus the bacterial inoculum (positive control), and culture medium plus Bio-AgNP and VAN (sterility control). The tests were performed in triplicate, with three replicates for each bacterial strain, and incubated at 36°C for 24h. After, 10 μL of resazurin 0.2% (Sigma ®, St. Louis, USA) was added to wells and the plate was incubated at 36°C for 2h.

For the MBC determination, aliquots were collected from each well that did not show a color change from blue to pink after adding the resazurin (Sigma ®, St. Louis, USA), being sown in plates containing BHI agar (Kasvi®, Italy), and incubated for 24h at 36°C. After, it was observed which concentrations present bactericidal activity, indicated by the absence of bacterial growth. The assays were performed in triplicate with three replicates for each strain.

Bacterial cell membrane integrity

The bacterial cell membrane integrity was monitored by the release of cell proteins to the medium supernatant. For this assay were used standard strains S. aureus ATCC 25904 and S. epidermidis ATCC 35984. The strains were treated with Bio-AgNPs, using the methodology proposed by Vasconcelos et al. [23]. For this, 1.5×10⁴ CFU.mL^-1 were treated with 1/2×MIC, MIC, 2×MIC, and 4×MIC of Bio-AgNPs and incubated at 37°C at times: 0h, 5min, 30min, 1h, 2h, and 4h. An aliquot of 1 μL of the treatments was added to BHI agar (Kasvi®, Italy) to confirm cell viability at different time points, being incubated at 37°C for 24h. Additionally, 150 μL were removed from each treatment at different time points and centrifuged at 2.500 × g for 5 min at 4°C. The concentration of proteins released from the cytoplasm was assessed in the supernatant using the Pierce BCA Protein Assay Kit (Sigma ®, St. Louis, USA). Optical density (OD) was read at 460nm as per the manufacturer's instructions using the POLARIS EE Spectrophotometer (Celer Biotecnologia S.A., Brazil). Bacteria untreated was used as a negative control and the experiments were carried out in duplicate.

Time-kill assay

The time-kill assay was performed according to the methodology described by Daniel- Jambun et al. [24] to evaluate the effect of Bio-AgNP on the growth curve bacteria strains. This assay was carried out with standard bacteria strains (S. aureus ATCC 25904 and S. epidermidis ATCC 35984) and two clinical isolates (SA3 and CoNS6). The bacterial inoculum (10⁶ CFU.mL^-1) was submitted to only BHI broth (Kasvi ®, Italy) (negative control), 2×MIC and 4×MIC of Bio-AgNP, plus BHI broth at 37°C. Thereafter, 100 μL were serially diluted in 900 μL of saline solution (NaCl 0.9%) at the times 0h, 10min, 20min, 30min, 2h, 4h, 6h, 8h, and 24h. These samples were transferred in duplicate to plates with Chapman Agar (Acumedia ®, Michigan, EUA) and incubated at 37°C for 24h. After, the colony-forming units (CFU) were counted and a time-to-death curve was constructed by plotting the log.CFU.mL^-1 versus negative control. The assay was performed in duplicate. Bactericidal activity was defined as a reduction of 99.9% (≥ 3 log10) of the total number of CFU.mL^-1 in the original inoculum [24].

Biofilm formation assay

The in vitro biofilm formation assay was performed in 96-well polystyrene microtiter plates [25]. The standard strains and clinical isolates suspension were prepared (1×10⁶ CFU.mL^-1) and 20 μL transferred to 180 μL of MH broth (Kasvi®, Italy) and incubated at 37°C for 24h. As a negative control, 200 μL of the culture medium was used. Then the contents of the plates were removed and washed with sterile saline solution (200 μL) to removed not added cells. The added cells were fixed with 99.8% methanol (200 μL) and stained with 0.5% of violet crystal (200 μL). The wells were washed with sterile saline and the dye was dissolved with 100% ethanol (200 μL). The optical density of biofilms was measured at 540nm using POLARIS EE Spectrophotometer (Celer Biotecnologia S.A., Brazil). The classification of the biofilms was made to the optical density of the negative control (ODc). Biofilms were classified as non-biofilm producer (OD ≤ ODc), weak (ODc < OD ≤ 2×ODc), moderate (2×ODc < OD ≤ 4×ODc), and strong (OD ≥ 4×ODc) [25]. The experiments were performed in triplicate.

Biofilm inhibition assay

The S. aureus ATCC 25904, S. epidermidis ATCC 35984, and clinical isolates (SA3 and CoNS6) were exposed to the different concentrations of Bio-AgNP before induction of biofilm formation, to determine if these concentrations would be capable of inhibiting or avoiding your establishment. For this, 20 μL bacterial suspension (1×10⁶ CFU.mL^-1) was added in 180 μL of MH broth (Kasvi®, Italy) with MIC, 2×MIC, and 4×MIC of Bio-AgNP into a 96-well polystyrene flat bottom microplate and incubated at 37°C for 24h. Controls used were: MH broth with evaluated concentrations of Bio-AgNP, MH broth without Bio-AgNP, and MH broth only. Then, the process of removal of the non-adhered cell, fixation, coloring, and staining of the biofilm was performed as mentioned previously. The results were expressed as a percentage of inhibition through the formula: 100 × (1 − (OD_treatment − OD_{negative control})/(OD_{positive control} − OD_{negative control})). Where OD_treatment is the OD of bacterial biofilm exposed to different concentrations of Bio-AgNP, OD_{negative control} is the OD of wells containing MH broth only, and OD_{positive control} is the OD of wells containing the biofilm untreated [26]. The experiments were performed in triplicate.

Biofilm eradication assay

The biofilm eradication assay was evaluated in vitro for S. aureus ATCC 25904, S. epidermidis ATCC 35984, and clinical isolates (SA3 and CoNS6) into a 96-well polystyrene flat bottom microplate. After the induced in vitro biofilm formation, 200 μL with MH broth (Kasvi®, Italy) with MIC, 2×MIC, and 4×MIC of Bio-AgNP were added and incubated at 37 ° C for 24h. Then, the process of removal of the non-adhered cell, fixation, coloring, and staining of the biofilm was performed as mentioned previously. The results were expressed as a percentage of inhibition through the formula: 100 × (1 − (OD_treatment − OD_{negative control})/(OD_{positive control} − OD_{negative control})). Where OD_treatment is the OD of bacterial biofilm exposed to different concentrations of Bio-AgNP, OD_{negative control} is the OD of wells containing MH broth only, and OD_{positive control} is the OD of wells containing the biofilm untreated [26]. The experiments were performed in triplicate.

Confocal laser scanning microscopy (CLSM) analysis

This assay was realized on establish biofilms of S. aureus ATCC 25904, S. epidermidis ATCC 35984, and clinical isolates (SA3 and CoNS6). For this, biofilm formation was previously induced in 6-well plates flat bottom and add 100 μL of inoculum (1×10⁶ CFU.mL^-1) in 900 μL of MH broth to cover a coverslip and incubated at 37°C for 18h. Subsequently, the biofilms were removed and treated with 2×MIC Bio-AgNP at the same conditions. A control treatment was also performed (without Bio-AgNP). After, the biofilms were washed with PBS 1%, cells were stained with SYTO 9 and propidium iodide, according to the manufacturer's recommendations (LIVE/ DEAD™ BacLight™ - Invitrogen, USA). Next, the assay was observed with a Leica TCS SP8 confocal laser scanning microscope at 100× magnification [27]. The experiments were performed in duplicate.

Statistical analysis

Statistical analysis was carried out by Two-Way analysis of variance (ANOVA) for time-kill and integrity of membrane assays. Dunnett’s post-test was conducted to identify significant differences between the treatments with concentrations of Bio-AgNP at different times evaluated. For the biofilm and antibiofilm were used One-way ANOVA and Tukey post-test to identify the difference between the treatments. For the analysis was used the GraphPad Prism 8.2.0 software was where values of p<0.05 were considered statistically significant.

Results

AgNPs aliquots were measured by absorbance at 440 nm in a UV-Vis spectrophotometer (Varian Cary 50 Probe®, Australia). The diameter and zeta potential of the Bio-AgNPs were determined by photon correlation spectroscopy using ZetaSizer Nano ZS. The Zeta potential (mV) of the bio-AgNPs used in this study was −32.9, and its size was 100 nm.

The antibiogram and MIC of VAN results are described in Table 1 and it was observed that the bacteria evaluated in our study present an important resistance profile to different antibiotics. Furthermore, the mecA gene responsible for resistance to β-lactam drugs was detected in all isolates evaluated, classifying them as MDR. The Bio-AgNP MIC values vary between 3.75 and 15 μL.mL^-1 and the MBC varying 7.5 and 30 μL.mL^-1 (Table 1). The MIC₅₀ and MIC₉₀ were 7.5 and 30 μL.mL^-1 for S. aureus and CoNS strains, respectively.

Table 1.

Resistance profile, mecA gene, in vitro biofilm formation, minimal inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) of Bio-AgNP against multi-resistant clinical isolates of Staphylococcus spp

Bacterial strains	Resistance profile	mecA gene	Biofilm ⁑	VAN	Bio-AgNP
				MIC (μg.mL-1)	MIC (μg.mL-1)	MBC (μg.mL-1)
ATCC 25904	*	-	+++	*	7.5	30
SA3	CEF; ERI; OXA	+	+++	8	7.5	30
SA4	CEF; ERI; OXA	+	+++	2	7.5	15
SA5	CEF; ERI; OXA; GEN	+	+++	2	15	30
SA6	ERI; OXA	+	+++	4	7.5	30
SA9	-	+	+++	16	7.5	15
ATCC 35984	*	-	+++	*	7.5	30
CoNS3	-	+	+++	4	7.5	15
CoNS5	ERI	+	+++	2	15	30
CoNS6	CEF; ERI; OXA; GEN	+	+++	2	3.75	7.5
CoNS16	CEF; ERI; OXA	+	+++	8	7.5	30
CoNS17	CEF; ERI; OXA	+	+++	8	7.5	15

CEF, cefoxitin; ERI, eritromicin; OXA, oxacillin; GEN, gentamicin; VAN, vancomycin; +, the presence of mecA gene; -, negative result; *, not applied; +++, strong biofilm formation in vitro; MIC, minimal inhibition concentration; MBC, minimal bactericidal concentration; SA, clinical isolate Staphylococcus aureus; CoNS, clinical isolate coagulase-negative Staphylococcus; ⁑, based on Stepanovic et al. [25] classification

The protein concentration increased significantly according to the concentration of Bio-AgNP, as described in Fig. 1. In the treatment with 2×MIC and 4×MIC (at 4h) the highest concentrations of total proteins were obtained for both bacteria. It was possible to observe that for both bacteria, there was cell growth in treatments with 1/2×MIC and MIC, at times 0 and 30 min only, and for the other treatments, there was no detectable growth. These results suggest that Bio-AgNP cause changes in membrane integrity, leading to cell content leakage.

Fig. 1.

Protein leakage by S. aureus ATCC 25904 and S. epidermidis ATCC 35984 after treatments with several concentrations of biogenic silver nanoparticles (Bio-AgNP). MIC: minimal inhibitory concentration; different letters (a, b, and c) mean the statistical difference between treatments (p<0.05)

In the time-kill assay, the 2×MIC showed bactericidal activity in ATCC 35984 after 6h of treatment, although for the CoNS6 the Bio-AgNP did not show bactericidal effect under the tested concentrations. For ATCC 25904 and SA3, the time of death was observed after 24h (Fig. 2). In the 4×MIC treatment, was observed that for all bacteria, the time of death cell was 6h, except for CoNS6 that was 24h. After 6h of exposure, the treatment with 4×MIC had a difference for all evaluated bacteria compared to the control and to 2×MIC there was no difference only for ATCC 35984.

Fig. 2.

Time-kill curve of multidrug-resistant isolates and standard strains of S. aureus and coagulase-negative Staphylococcus treated with biogenic silver nanoparticles (Bio-AgNP). SA3: S. aureus clinical strain; CoNS6: coagulase-negative Staphylococcus clinical strain; ATCC 25904: S. aureus ATCC 25904; ATCC 35984: S. epidermidis ATCC 35984; MIC: minimal inhibitory concentration. 2×MIC Bio-AgNP = 15 μg.mL^-1 for ATCC 25904, ATCC 35984, and SA6; 2×MIC Bio-AgNP = 7.5 μg.mL^-1 for CoNS; 4×MIC Bio-AgNP = 30 μg.mL^-1 for ATCC 25904, ATCC 35984, and SA6; 4×MIC Bio-AgNP = 15 μg.mL^-1 for CoNS. *p<0.05 in comparison to control (untreated)

All bacteria evaluated were classified as biofilm-strong formers (Fig. 3). In the biofilm inhibition assay, it is possible to observe that the application of treatments with Bio-AgNP significantly reduced the formation of biofilms in vitro. However, no significant differences were observed between the treatments used (MIC, 2×MIC, and 4×MIC), as demonstrated in Fig. 4.

Fig. 3.

In vitrobiofilm formation in multidrug-resistant isolates of S. aureus and coagulase-negative Staphylococcus

Fig. 4.

Inhibition of biofilm by biogenic silver nanoparticles (Bio-AgNP) against multidrug-resistant isolates and standard strains of S. aureus and coagulase-negative Staphylococcus. SA3: S. aureus clinical strain; CoNS6: coagulase-negative Staphylococcus clinical strain; ATCC 25904: S. aureus ATCC 25904; ATCC 35984: S. epidermidis ATCC 35984; MIC: minimal inhibitory concentration. *p<0.05 in comparison to control (untreated strain); 2xMIC Bio-AgNP: 15 μg.mL^-1 for ATCC 25904, ATCC 35984 and SA6, 7.5 μg.mL^-1 for CoNS; 4xMIC Bio-AgNP: 30 μg.mL^-1 for ATCC 25904, ATCC 35984, and SA6, 15 μg.mL^-1 for CoNS. *p<0.05 in comparison to control (untreated)

In the biofilm eradication assay, for S. aureus ATCC® and SA3, was no observed significant difference between the 2×MIC and 4×MIC treatments, and eradication percentages ranged from 63.3% to 94.9% after treatments (Fig. 5). For S. epidermidis ATCC® 35984, the eradication ranged from 60.89% to 78.84% after the application of treatments with Bio-AgNP, with a significant difference being observed between the lowest concentrations applied (MIC and 2×MIC) when compared with 4×MIC. For the CoNS6 isolate, MIC was not able to eradicate the biofilm; however, 2×MIC and 4×MIC eradicated 51.22% and 70.75%, respectively.

Fig. 5.

Eradication of biofilm by biogenic silver nanoparticles (Bio-AgNP) against multidrug-resistant isolates and standard strains of S. aureus and coagulase-negative Staphylococcus. SA3: S. aureus clinical strain; CoNS6: coagulase-negative Staphylococcus clinical strain; ATCC 25904: S. aureus ATCC 25904; ATCC 35984: S. epidermidis ATCC 35984; MIC: minimal inhibitory concentration. *p<0.05 in comparison to control (untreated strain); 2xMIC Bio-AgNP: 15 μg.mL^-1 for ATCC 25904, ATCC 35984 and SA6, 7.5 μg.mL^-1 for CoNS; 4xMIC Bio-AgNP: 30 μg.mL^-1 for ATCC 25904, ATCC 35984 and SA6, 15 μg.mL^-1 for CoNS. *p<0.05 in comparison to control (untreated); different letters represent significant differences between treatments (a, b)

The CLSM assays were performed to visualize the dead cells in the biofilm eradication test, using the 2xMIC concentration of Bio-AgNP, since it was the most efficient concentration in the in vitro assays and was not significantly different from the highest used concentration (Fig. 6). For this, the SYTO9 brushes the viable cells, emitting green fluorescence, while the damaged/dead cells are stained by propidium iodide, emitting red fluorescence. From these results, it is possible to observe that the dead cells are visibly higher after treatment with Bio-AgNP when compared to controls (without Bio-AgNP) in standard strains and MDR isolates, which was observed in eradication assays proposed by Halicki et al. [26].

Fig. 6.

Confocal laser scanning microscopy of standard strains and clinical isolates of S. aureus and coagulase-negative Staphylococcus and their biofilms after treatment with 2xMIC Bio-AgNP. SA3: S. aureus clinical strain; CoNS6: coagulase-negative Staphylococcus clinical strain; ATCC 25904: S. aureus ATCC 25904; ATCC 35984: S. epidermidis ATCC 35984; green: live cells; red: dead cells; A ATCC 25904 (without Bio-AgNP); B ATCC 25904 2xMIC Bio-AgNP; C ATCC 35984 (without Bio-AgNP); D ATCC 35984 2xMIC Bio-AgNP; E SA3 (without Bio-AgNP); F SA3 2xMIC Bio-AgNP; H CoNS 6 (without Bio-AgNP); I CoNS 6 2xMIC Bio-AgNP

Discussion

According to WHO [7], research and development of new drugs against MRSA are a high priority and we report for the first time the activity of this Bio-AgNP against CoNS and MRSA clinical strains. The evaluation of Bio-AgNP in vitro for the control of these pathogens is promising and necessary, as demonstrated in our study and other recent research [6–8, 27–30].

These nanoparticles have the advantage of being synthesized by green chemistry, are ecologically friendly, and different studies describe their antibacterial activity. In the time-kill assay, the 2×MIC (15 μg.mL^-1) was able to kill all bacteria evaluated after 24h of treatment, except the isolate CoNS6 (7.5 μg.mL^-1). The 4×MIC (30 μg.mL^-1) and 2×MIC (15 μg.mL^-1) concentrations were bactericidal in 6h of treatment for all strains in the study. The major of studies observed bactericidal activity in at least 24h of exposure [31–33] and a few studies evaluate the Bio-AgNP against CoNS [34].

According to the in vitro assay, the bacterial cell protein release is dose-dependent, when we increased the concentration of Bio-AgNP increased the protein leakage, indicating that one of the possible sites of action of the nanoparticle may be at the cell membrane. Similar results were related by Yu et al. [15], where higher concentrations of Bio-AgNP (15 to 63 μg.mL^-1) lead to the damage being more accentuated. Other studies demonstrate changes in membrane potential and cell membrane integrity in S. aureus and MRSA when treated with AgNP (6–43 μg.mL^-1) at different times [7].

For Shatan et al. [6], nanoparticles probably do not enter the bacterial cell rapidly; however, their accumulation can lead to cell wall rupture and favors the entry of particles in the cytoplasm. Thus, a longer exposure time is necessary for Bio-AgNP to exert its antibacterial activity. Strong evidence indicates that AgNP can adhere to cell membranes, accumulate, and negatively charge the cell surface, altering its permeability, and can interact with DNA, proteins, lipid compounds, and cellular constituents that present phosphorus [6, 35]. In our study, the Bio-AgNPs induced the bacteria cells’ death after 6 h.

The Bio-AgNP demonstrated the ability to reduce and eradicate in vitro biofilm in standard strains and MDR isolates at concentrations of 3.75 to 30 μg.mL^-1. In addition, biofilm eradication results are confirmed through the CLSM assays (Fig. 6). Was possible to observe a higher percentage of dead cells in the biofilm eradication assay with Bio-AgNP when compared with the control treatment (without Bio-AgNP), where a large amount of green was observed, represent the viable cells of the biofilm. Our results are very promising since biofilms are considered an important virulence factor, mainly related to the use of invasive medical devices and pneumonia associated with mechanical ventilation and chronic wounds [31, 36].

Furthermore, based on results described in the literature, there is strong evidence that Bio-AgNP does not lead to the development of resistance in S. aureus after successive exposures, indicating a good potential for application against these pathogens, including MDR [36]. Therefore, the Bio-AgNP evaluated in our study demonstrates in vitro activity against clinical isolates of MRSA and CoNS MDR and we consider that nanoparticles, in addition to having the advantage of being obtained through an environmentally friendly synthesis, can be potential allies against Staphylococcus spp. MDR pathogens.

Author contributions

DDH and KFC conceived and designed the study; KFC, DTFA, MOG SOA, and ACPSN carried out experimental work; DDH, KFC, ACPNS, LAP, and TL carried out the analysis and interpretation of data, analysis, and technical support; KFC wrote the manuscript; DDH and ACPN supervised the work and reviewed the manuscript.

Funding

This work was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES, http://www.capes.gov.br/) - Finance Code 001 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, http://www.cnpq.br/) which provided research (DDH) and scholarship (KFC, MOG, SOA, DTFA, and ACPSN).

Declarations

Competing interests

The authors declare no competing interests.