Nano silver combined with antibiotic to help enhance the bactericidal effect

The ability of microorganisms to create resistance faster than to create new and effective antibiotics; therefore, it is important to develop new antibiotic agents and treatments to control bacterial infections. An alternative to this worldwide problem is to use nanomaterials with antimicrobial properties.Nano silver (AgNPs) have been extensively studied for their antibacterial effects on various organisms. In this study, the synergistic antibacterial effects of AgNPs and common antibiotics were evaluated in Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration of AgNPs was 10–12 μg / ml in all strains tested, regardless of their varying susceptibility to antibiotics.

Interestingly, a synergistic antimicrobial effect was observed when combining AgNPs and kanamycin according to the fractional inhibitory concentration index, FICI: <0.5), an effective additive when combining AgNPs and chloramphenicol (FICI: 0.5 to 1), while no effects were found with AgNPs combining β-lactam antibiotics. Cell flow measurements and TEM analysis showed that the sublethal concentration of AgNPs (6–7 μg mL -1) alters the bacterial membrane potential and induces superstructure damage, increasing membrane permeability. cell. No chemical interactions between AgNP and the antibiotic were detected.

We propose a test-supported mechanism of action in which the synergistic effect of antimicrobials promoting synergy depends on their specific goal, facilitated by substitution. membrane modifications made by AgNPs. Our results provide a deeper understanding of the synergistic mechanism of AgNP and antibiotics, which are aimed at effectively fighting infection caused by antibiotics, especially multidrug-resistant microorganisms, to minimizes the current crisis due to antibiotic resistance.

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Introduce

Infectious diseases caused by pathogenic bacteria are one of the most common causes of death worldwide and a frequent health risk in all countries [1]. Today, the burden of infectious diseases on health, economics and other social aspects is so complex that worldwide costs cannot be estimated [2]. Current antibiotic therapies have significant disadvantages, such as limited diversity, antagonistic interactions and the effect of incomplete antibiotic treatments leading to drug-resistant microorganisms, among many other methods [3].

Multidrug-resistant bacteria are one of the most serious public health problems worldwide [4], and new strains of resistant bacteria regularly appear to diminish the effectiveness of current treatments. leading to serious public health risks [5, 6]. It also negatively affects certain areas of human activity, such as agriculture, aquaculture and veterinary [5, 7]. Unfortunately, the evolution of multi-resistant bacteria outpaced the development of new antibiotics; therefore, the creation of new and effective antimicrobial treatments is important [8].

Strategies for maintaining the antibiotic efficacy have been recommended [5]. One of the alternatives to fight against multidrug-resistant organisms is to use nano antibiotics (nanomaterials with antibacterial properties [9, 10]). Due to their antiviral and antibacterial properties, silver nanoparticles (AgNPs) are the most promising nano antibiotics available today [11 – 15]. Advantages of AgNPs are a generalized mode of action against various pathogens, such as viruses, bacteria and fungi; as well as their antibacterial effect does not depend on the susceptibility of the microorganism to conventional antibiotics, including the flow pump and biofilm formation [16, 17], hence AgNPs. can overcome microbial resistance to conventional antibiotics [18]. Furthermore, the use of AgNPs has advantages other than their antiviral and antimicrobial activity. AgNPs can be synthesized using easy, inexpensive and environmentally friendly methods, and they can be produced by chemical or biosynthesis processes [19–21]. AgNPs showed a way of acting in multiple levels on bacterial cells to influence metabolic processes: A) disrupting cell walls and membranes and increasing cell permeability [22-25]; B) Nano silver ingress and intracellular damage disrupt metabolic pathways [24, 26, 27]; C) damage to biological molecules (DNA, proteins) [25]; and D) oxygenation reaction [28-30]. For more recent reviews see [31, 32].

Recently, improvements in antibacterial activity – synergistic effects – have been reported when AgNPs are combined with several antibiotics, such as ampicillin, amoxicillin and chloramphenicol [33–38]. In contrast, reports of antagonistic interactions between AgNPs and amoxicillin or oxacillin have been reported [37]. Studies focusing on the mechanism of action of the NPs-antibiotic combination have suggested that the improvement in antimicrobial activity may be due to their chemical interactions, however, the underlying molecular mechanism of effects, synergistic or antagonistic, still need to be clarified [34, 36, 39].

In the presence of conflicting reports, we are interested in assessing the antibacterial activity of nano-silver in combination with the antibiotic of different cell targets and, therefore, different mechanisms of action. together. We chose chloramphenicol (Cm), kanamycin (Km), ampicillin (Amp), aztreonam (Azm) and biapenem (Bpm). The enhanced effectiveness of the AgNPs antibiotic combination is characterized by various analytical techniques to elucidate the antimicrobial mechanisms involved. Since synergy is strongly associated with antibacterial conjugate therapies, we have explored both Gram-positive and Gram-negative bacteria. We can determine that AgNPs alter the integrity and potential of the cell membrane, increase cell permeability and facilitate intracellular access to antibiotics,

Materials and methods

Properties of silver nanoparticles (AgNPs)

Argovit AgNPs are taken from Vector Vita Ltd (Novosibirsk, Russia). They are stabilized using polyvinylpyrrolidone (PVP). Silver nanoparticles are characterized by physical and chemical methods (Figure S1). In summary, surface plasmon resonance is characterized by spectral UV-Vis (Multiskan Go, Thermo Scientific) measurements in the 270 to 600 nm range. The morphology and chemical composition of AgNPs were examined in TEM (Jeol JEM 2100). In addition, elemental composition analysis (EDX) was performed to confirm the presence of silver. Their mean hydrodynamic diameters and nano-silver stability were measured using dynamic light scattering (DLS) method using nano Zeta sizer [40].

Strains and culture conditions of bacteria

Tests were performed on four strains of bacteria. Gram-negative: Escherichia coli DH5α (non-pathogenic) and Salmonella enterica serovar Typhimurium ATCC SC14028 (S. Typhimurium) (pathogenic); Gram-positive: Staphylococcus aureus (clinical isolate, pathogenic) and Bacillus subtilis (non-pathogenic), from collection kept at Centro de Nanociencias y Nanotecnologia – Universidad Nacional Autónoma de Mexico. For the experiments, all bacteria were cultured in Muller-Hinton medium (MH). For MH plates, 1.5% agar was added. The cultures were incubated for 24 hours, 37 ° C and 180 rpm.

Antimicrobial agent

Antibiotics: chloramphenicol (Cm), kanamycin (Km), biapenem (Bpm), and aztreonam (Azm) were taken from Sigma-Aldrich (USA), and ampicillin (Amp) from Duchefa Biochemie (USA). The selected antibiotic concentration range is based on protocol guidelines CLSI M100 [41] and Vazquez-Muñoz, et. al., 2017 for AgNPs [42]. Minimum inhibitory concentrations (MIC) were determined for AgNPs and all antibiotics used in this study (Table 1). The sublethal concentrations for the combinatorial effects assays were selected based on MIC (Table S1).

Antibacterial activity of AgNPs, AgNO3 and antibiotic

Minimum inhibitory concentrations (MIC) and sublethal concentrations were determined for nano silver, AgNO 3 and antibiotics. MIC was evaluated according to the Clinical Laboratory Standards Institute M07-A9 protocol [43], with some modifications. The concentration of Sublethal was determined optically by UV-Vis spectroscopy, at a wavelength of 600 nm. As control, for each concentration of AgNPs and AgNO 3 tested, a solution without inoculum is used.

Antibiotic treatment method combined with AgNPs

The combined activity of the sublethal concentration of AgNPs (6 μg.mL -1) with different antibiotics was evaluated by microdilution method. For each antibiotic, the concentration below the lethal level used was half the MIC (Table S1). In the control, the bacteria were exposed separately to the lethal concentrations of AgNPs and the respective antibiotics. Cells were incubated under standard laboratory conditions (37 ° C and 180 rpm), in 96-well plates for 24 hours. Inhibition of bacterial growth was determined by spectroscopy at 600 nm. To determine the interaction effect of antibiotic and nano-silver, a fractional inhibitory concentration index (FICI) modified by a Ratio [44], was used. The FICI suggested that a pairing combination of agents could cause either an inhibitory effect greater than their total effect alone (synergy; FICI ≤0.5), or less than their total effect. single (antagonistic; FICI> 4) [44]. For this study, an additive effect was considered when FICI values ​​were> 0.5 and ≤1. FICI was determined by calculating the percentage inhibition of bacterial growth for AgNP and antibiotics according to the formula:

Interaction characteristics between nano silver-antibiotic by optical analysis

AgNPs-antibiotic combination treatments were analyzed by Fourier Transformer infrared spectroscopy (FT-IR) to evaluate potential chemical interactions between AgNPs and antibiotics. Combination treatments were incubated in deionized water under standard conditions and then freeze-dried. Finally, the samples were centrifuged and the pellets mixed with potassium bromide (KBr). FT-IR analysis was performed on the Nicolet 6700’s FT-IR spectrometer, Thermo Scientific ®, in the range 4000 to 400 cm -1.

Dynamic light scattering analysis (DLS) was performed to assess the stability of AgNPs on antibiotic exposure based on changes in aggregation (hydrodynamic diameter) and charge (potential Z). AgNPs (50 μg.mL -1) mixed with antibiotic (100 μg.mL -1) diluted in deionized water and measured at 0 hours and 24 hours, under culture conditions (37 ° C, 180 rpm / minute). DLS analysis was performed in Zetasizer Nano ZS (Malvern) system.

Analysis of superstructures by transmission electron microscopy (TEM)

Effects of AgNP on bacterial cell wall structure and their bioaccumulation capacity were assessed by electron microscopy. E. coli, S. aureus, S. Typhimurium and B. subtilis was exposed to lethal concentrations of AgNPs and AgNO3 (6 and 4 μg.mL-1, respectively), and incubated under standard conditions. The bacterial cells were then centrifuged, fixed with glutaraldehyde (2%), and then immobilized with OsO 4 (1%). The samples were dehydrated and soaked into Spurr’s resin. Polymerized samples were cut into sections (100 nm) in microscopic PowerTome X (RMC Boeckeler). The sliders are mounted in 300 mesh copper meshes covered with threaded carbon (Ted Pella). Superstructure analysis was performed with TEM Jeol JEM-2010 (CNyN, UNAM) and TEM Jeol JEM-1230 at 80 KeV (Department of Pathology, National Heart Institute). On the other hand, the interaction between AgNPs-antibiotic was assessed by mixing AgNPs and antibiotics separately for 24 hours. AgNPs (50 μg.mL -1) mixed with antibiotic (100 μg.mL -1) diluted in deionized water. One drop of the mixture was placed in a 300 mesh copper mesh covered with threaded carbon (Ted Pella), dried and observed with a TEM.

Effect of nano silver on the potential and permeability of bacterial cell membranes

The effect of AgNPs on cell membrane polarization was evaluated according to the procedure proposed by Novo and Perlmutter [45]. (In summary, each strain of E. Coli, S. Typhimurium, S. Aureus and B. Subtilis) was cultured under standard conditions and adjusted to 1 x 10 6 cells per mL in sterile 1x PBS. Bacterial cultures were exposed to 10 μg.mL -1 of AgNPs or AgNO 3 and stained with 18 μM DiOC6 (Sigma-Aldrich, USA). Positive controls were exposed to 50 μM CCCP (Sigma-Aldrich, USA). The cells were incubated at room temperature for 1 hour and analyzed in an Attune flow meter (Thermo Scientific), with a laser emitted at a wavelength of λ = 488 nm. Fluorescence is collected using green and red channels. Front dispersion, lateral dispersion and fluorescence data were obtained by amplifying logarithmic signals. To assess membrane permeability, each strain was cultured under the same conditions and exposed to AgNPs or AgNO3 as described above. Cells were stained with the cell viability reagent alamarBlue (Thermo Scientific) and 75 nM propidium iodide. Cells were incubated at 37 ° C for 1 hour and analyzed by flow measurement with purple and blue lasers emitted at λ = 405 and 488 nm, respectively. Fluorescence was recorded with the VL3 and BL3 channels as described previously.

Result

Properties of silver nanoparticles (AgNPs)

Argovite AgNP formula used in this study contains 1.2% metallic silver stabilized with 18.8% polyvinylpyrolidone (PVP) in water (Vector-Vita Ltd). The UV-Vis profile of AgNPs showed a peak at 400 nm, which corresponds to typical surface plasmon resonance of AgNPs. TEM images show that AgNPs are spherical and that their mean diameter is 35 ± 15 nm. Elemental composition analysis (EDX) confirmed the presence of silver. Their average hydrodynamic diameter is 70.4 ± 0.5 nm, and their zeta potential value is -18 ± 1.1 mV. (Figure S1). AgNPs concentration is calculated by the content of metallic silver.

Minimum inhibitory concentrations (MIC) of AgNPs, AgNO 3 and antibiotics

Both Gram-negative and Gram-positive strains were incubated with different antibiotic concentrations to obtain minimum inhibitory concentration (MIC) and sublethal concentration. E. coli, S. Typhimurium, B. subtilis and S. the aureus is inhibited at between 10 and 12 μg.mL-1 of AgNP and 6–7 μg.mL-1 of AgNO 3. In contrast, all strains showed significantly different sensitivities to the antibiotics tested, with MICs ranging from 0.05 to 32 μg.mL -1 (Table 1). According to the standard clinical and laboratory Institute of Standars (CLSI) in the M100-S17 guidelines [46], our results show S. aureus resistance is only Km (32 μg.mL -1), while B. subtilis is resistant to Cm and Azm (16 and 32 μg.mL-1 respectively). E. coli and S. Sensitivity to typhimurium was detected for all antibiotics tested (<2 μg.mL -1). Interestingly, the results for MBC and MIC were the same for all bacterial strains in AgNPs and AgNO 3 treatments (data not shown).

Antimicrobial activity of AgNPs combination antibiotic treatments

When the MIC value was determined for all antimicrobial agents, lethal concentrations of nano-silver combination antibiotic treatments were evaluated to determine synergistic effects. Sublethal concentrations used for nano-silver were 6 μg.mL-1, and half MIC for each antibiotic (Table S1). In the control, the bacteria were individually exposed to lethal concentrations of AgNPs and antibiotics.

The AgNPs-Cm combination induced around 50% growth inhibition compared to E. coli, S. Typhimurium, and S. aureus, while AgNPs-Km caused significant growth inhibition on the same strain (about 95%) (Figure 1). A fractional inhibition concentration index (FICI) was calculated for each silver-antibiotic nanoparticle [44]. For this study, synergy was reported in the cases as FICI ≤0.5, an adverse effect when FICI values ​​were between 0.5 and 1, and antagonistic for FICI> 4. No effects were considered for FICI> 1 to <4. Interestingly, statistical differences were observed when combined with either AgNPs Km or Cm in E. coli, S. Typhimurium and S. aureus, respectively (Figures 1 and 2). However, the synergistic effect was observed only for the combinations Km while only the resonance effect was determined for AgNPs-Cm. No effects were observed when combining non-AgNPs with β-lactam antibiotics to any tested microorganisms, nor any treatment in B. subtilis (Figure 1).

Table 2 Fractional inhibitory concentration index (FICI) of antibiotic and AgNPs combination treatments.

Spectral characteristics DLS and FT-IR of antibiotic and AgNP treatments combined

To determine whether there is a direct interaction between nano silver and an antibiotic, we performed FT-IR and DLS analyzes of different antibiotics and AgNP, in single treatments. odd or combined. The FT-IR infusion configurations of the AgNPs combination antibiotic treatments have characteristic peaks of both PVP-AgNPs and standard antibiotics (Figure S2). Therefore, transmission spectroscopy analysis showed no evidence of a covalent chemical bond between nano-silver and an antibiotic.

On the other hand, DLS analysis shows that Km and Amp have an effect on AgNPs size and charge (Figure S3). The changes in size and charge may be due to an electrostatic interaction between the AgNPs and the antibiotic, where the Km and Amps can either be absorbed around the AgNPs or cause their agglomeration. In some cases, these differences were evident with a color change in the antibiotic-AgNPs mixture (data not shown). To clarify the DLS results, we analyzed the size and agglomeration state of the AgNPs when combined with Km and Amps by transmission electron microscopy (TEM). In accordance with the DLS data, AgNPs aggregation was observed for the mixture of AgNPs-Km and AgNPs-Amp, while no aggregation was observed when combining AgNPs with Cm or Azm (Figure S4).

Effect of nano-silver on cell metamorphosis

To compare the effect on membrane integrity of NPs, strains were treated with concentrations of AgNPs and AgNO3 below lethal levels, and cell morphology was shown using TEM (Figure 5). . Compared to untreated cells, both AgNPs and AgNO3 disrupted treatments E. coli and S. The typhimurium cell membrane, which can be seen due to loss of integrity, shows a direct effect of silver ions on the membrane stability (Figures 2 and 3). The interesting thing is for B. subtilis and S. aureus (both Gram-positive), exposed to levels of AgNPs or AgNO3 below lethal levels, caused no apparent damage (Figures 4 and 5).

For E. coli, S. Typhimurium and S. aureus, the sublethal concentration of the AgNPs-Km and AgNPs-Cm combination treatments showed synergistic and resonant effects respectively (Table 2, Figure 1). As expected, TEM analysis under these two combined treatments showed significant cell damage confirmed MIC tests and indicated bacterial cell death (Figure S5).

Effect of nano-silver on the membrane potential and permeability

Gram-negative membrane lesions may explain the bactericidal effects of AgNPs when combined with Km or Cm. However, for S. aureus no structural changes were observed but a significant resonance and resonance effect was detected for Km and Cm, respectively. Previous reports have suggested that AgNPs and AgNO 3 cause depolarization and membrane instability leading to the microbiological effects of AgNPs and AgNO3 [45]. To evaluate the depolarization and permeability of membranes, we analyzed bacterial cells treated with AgNPs or AgNO 3 concentrations below lethal levels by flow cytometry. Interestingly, AgNPs and AgNO 3 depolarized bacterial membranes after 30 minutes of exposure, and these effects were not clear for Gram-negative and Gram-positive bacteria (Figure 6). Furthermore, permeability was also altered due to changes in the membrane potential in cells exposed to AgNPs and AgNO3 in all tested strains (Figure 7).[ 47 ].

Discussion

In accordance with the previous comparative analysis, the minimum inhibitory concentrations (MIC) of nano silver and AgNO 3 were similar in all strains tested, regardless of structural or biogenic difference. of them, with most MIC values ​​in the 6–12 μg.mL-1 range, and the same NPs concentration determined for the minimum bactericidal concentration (MBC) (Table 1) [42]. In contrast, antibiotic susceptibility may vary between strains.

TEM analysis showed that nano silver significantly affected cell membrane integrity of Gram-negative bacteria such as E. coli and S. Typhimurium (Figs (Figures 22 and 3) .3). However, in the treatment on Gram-positive bacteria (S. Aureus and B. Subtilis), no disruption of the cell wall was observed (Figs (Figures 44 and 5) .5). On the other hand, concentrations of AgNPs below the lethal level depolarized the membrane in all of the bacteria analyzed, resulting in increased cell permeability (Figure 6), regardless of differences in cell wall composition. between Gram-positive and Gram-negative bacteria [23, 25, 36, 48, 49]. This increase in membrane permeability can enable the antibiotic to enter the cell, allowing for a more effective intracellular antibiotic (Figure 7).

Silver nanoparticles and antibiotic combination treatments have been studied before but no mechanism of action has been established because different phenotypes have been detected between studies [22, 33, 37] . Furthermore, the effect of the antibiotic activity of AgNPs may differ in different organisms [50]. This difference found in AgNPs-synthetic antibiotic can be explained by different factors: the stabilizer used and its relative concentration, which may affect the interoperability between AgNPs and antibiotics [34, 49, 51]. AgNPs functionalized with PVP showed better synergistic antibacterial activity compared with those stabilized with either citrate or SDS [51]. Furthermore, interactions of AgNP stabilized with citrate and ampicillin have been reported [34], while AgNP stabilized with PVP did not show such interaction (this work), which indicates that the intention plays a central role in this type of interaction and at the same time with an antimicrobial (synergistic, additive or ineffective) interaction.

In this study, AgNPs-Km combination showed a synergistic effect on bacteria growing in E. coli, S. Typhimurium and S. aureus; while the AgNPs-Cm combination exhibited an additive (but still significant) effect on the same strains (Figures 1 and 2). Panacek et al. (2015) reported synergistic interactions between antibiotics with different modes of action and AgNPs, including AgNPs-β-lactam antibiotics [49]. However, it is important to note that E. The coli strain used in this study showed resistance to ampicillin even without AgNPs treatment, and this ampicillin resistance was overcome by the combination of AgNPs-ampicillin. A similar effect was found for a clinically isolated S. aureus strain, which showed resistance to Km, however, when combined with AgNPs-Km treatment, we also observed a synergistic effect (Fig. 1 and Ban 2). The synergy between nano silver and antibiotic was previously thought to be due to the chemical bond between the sulfur antibiotic group and AgNPs [36, 38], but no conclusive experimental evidence has been provided to support this. support this hypothesis. Deng et al. have reported synergistic effects and interactions between AgNPs and some antibiotics (enoxacin, kanamycin, neomycin and tetracycline). They proposed that this interaction promoted increased release of Ag ions, while also increasing inhibition of bacterial growth in Salmonella sp. [34]. However, our results showed no change in FT-IR experiments showing no covalent chemical interaction between nano silver and the antibiotics tested (Figure S2). For DLS analysis, superficial charge changes detected on AgNP combined with Km or Amp indicate electrostatic interaction. In addition, the AgNPs-Km combination also showed an increase in the hydrodynamic ratio (Figure S3). In the TEM image, AgNPs-Km or AgNPs-Amp aggregation is observed (Fig. S4). However, no direct correlation was found between the enhanced antimicrobial effect and changes in the interaction, size or charge of AgNPs. Therefore, it does not appear that these features are necessary for the bactericidal effects of the combined treatments. Interestingly, consistent with other studies, AgNPs alter membrane integrity and increase cell permeability (Figures 6 and 7) [15, 24, 25].

Here, we propose a mechanism of action of nano silver-antibiotic combination treatments, in which AgNPs destabilize the bacterial cell membrane, promote antibiotic endoplasm in the cell and at the same time. with microbicidal activity (Figure 8).

For those active inside the cell, either a synergistic or a synergistic effect can be observed since AgNP facilitates the invasion of the antibiotic cell and promotes access to their target ( for Km and Cm, ribosome) [52]. In contrast, β-lactam antibiotics do not synergize with AgNPs because these antibiotics work by affecting the integrity of the bacterial cell wall, so their effect is unaffected. by changes in the cell permeability and cell entrance of the antibiotic.

Our model is also supported by Deng’s findings, in which four intracellular antibiotics show synergy when combined with AgNPs [34]. Additionally, a recent report describes the enhanced activity of ciprofloxacin and gentamicin, among others, with both synergistic and synergistic effects, when applied in combination with AgNPs [48]. It is important to emphasize that the enhancement effect in antibiotic-resistant strains is higher than in antibiotic-sensitive strains, which can be explained by the model proposed in our study, in AgNP affects the cell membrane and cell wall integrity is conducive to the antibiotic effect, leading to a “restored” susceptibility to some resistant strains [48].

The physiological mechanism of the synergistic effect of nano silver and the antibiotic combination is explained here with experimental support. Our results help shed light on how the synergistic effects of these combinations occur. According to our experimental data, the enhancement of the antimicrobial activity of the antibiotic is due to the effect of AgNPs on the cell structure, not by the direct interaction with the AgNPs antibiotic.

Understanding how antibiotics, nano-silver, and combination therapies work can help predict more viable treatments and improve or design new, more effective treatments. Even if some aspects of the mechanism of action remain unknown, our results provide a more effective way to fight infectious diseases. The enhancement of antibacterial activity due to the nano silver antibiotic combination will allow the use of antibiotics that are no longer used because of drug-resistant bacterial problems, providing additional therapeutic possibilities in the care areas. health care, veterinary medicine and agriculture. Therefore, nano antibiotics have a potential impact on social and economic problems, as they can help mitigate the current crisis caused by antibiotic resistance.

Additional information

Figure S1

Silver nanoparticle properties.

A) UV-Vis profile. B) TEM image of AgNPs. C) The mean diameter of the AgNPs. D) Zeta potential analysis.

Figure S2

FT-IR analysis of AgNPs, antibiotics and combination therapies.

A) Ampicillin (Amp); B) Aztreonam (Azm); C) Kanamycin (Km); and D) Chloramphenicol (Cm). Transmission spectrum of AgNP antibiotic combinations is similar to that of AgNP antibiotic and antibiotic combinations.

Figure S3

DLS analysis of antibiotic and AgNP treatments combined at 0 (red) and after 24 hours of incubation (green).

The charge (A) and the size (B) of the AgNPs are different for each antibiotic

.

Figure S4

TEM images of antibiotic and AgNP treatments in combination.

A fusion effect was observed for the combination of Km + AgNPs and Amp + AgNPs (marked with arrows). Pictures were taken after 24 hours of incubation.

Figure S5

TEM images of bacterial cells exposed to concentrations of AgNPs and antibiotics in sublethal.

E. coli, S. Typhimurium, S. aureus and B. subtilis was exposed to the combination treatments for 24 hours. The representative image is displayed.

Table S1

Antibiotic additive concentration (μg.mL -1).

Reference source: Enhancement of antibiotics antimicrobial activity due to the silver nanoparticles impact on the cell membrane

R. Vazquez-Muñoz, Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing,^#1,2 A. Meza-Villezcas, Formal analysis, Investigation, Methodology, Validation, Writing – review & editing,^#1,2 P. G. J. Fournier, Formal analysis, Investigation, Methodology,² E. Soria-Castro, Formal analysis, Investigation, Methodology,³ K. Juarez-Moreno, Formal analysis, Investigation, Methodology,¹ A. L. Gallego-Hernández, Formal analysis, Investigation, Methodology, Writing – review & editing,⁴ N. Bogdanchikova, Conceptualization, Formal analysis, Resources,¹ R. Vazquez-Duhalt, Conceptualization, Formal analysis, Supervision,¹ and A. Huerta-Saquero, Conceptualization, Formal analysis, Funding acquisition, Investigation, Supervision, Writing – review & editing^1,*