Nano silver combined with antibiotics kill Acinetobacter baumannii multidrug-resistant bacteria

Acinetobacter baumannii resistance to carbapenem antibiotics presents a serious clinical challenge. As a newly developed technology, nano silver (AgNPs) exhibit some excellent properties compared to older treatments and are a candidate for fighting A. baumannii infection. However, its mechanism of action is still unclear. In this study, we combined AgNPs with antibiotics to treat carbapenem-resistant A. baumannii (aba1604). Our results showed that the single AgNPs completely inhibited A. baumannii growth at 2.5 µg / mL. Treatment with AgNP has also shown synergistic effects with polymixin B and rifampicin antibiotics, and side effects with tigecycline. In vivo, we found that the AgNPs-antibiotic combination resulted in better survival rates in A. baumannii-infected mouse peritonitis models compared with single drug treatment. Finally, we used strains of Escherichia coli that target different antagonistic RNA to elucidate the synergistic mechanism involved in bacterial responses to AgNP and antibiotics.

(Copyright by NanoCMM Technology)

Introduce

Acinetobacter baumannii is an infectious pathogen that presents serious clinical challenges. A. baumannii is particularly associated with infections acquired in hospitals such as pneumonia, blood sugar, abdomen, central nervous system, urinary tract, and skin and soft tissue infections. 1 A. baumannii can develop antibiotic resistance through a number of mechanisms; 2 in particular, this bacterium is often resistant to carbapenems. 3 An increasing number of carbapenem-resistant A. baumannii strains have been reported worldwide. 4 The majority of such bacteria are widely resistant to drugs, possibly including resistance to carbapenems and all other antibiotics except polymyxin and tigecycline. 5 Polymyxin B is effective against drug-resistant A. baumannii, but systemic use carries the risk of toxicity, mainly nephrotoxicity and neurotoxicity. 6, 7 A. baumannii infections are common in patients with severe infections, and are often accompanied by other bacterial and / or fungal infections. 8 Patients infected with drug-resistant A. baumannii have a high mortality rate. 9 Therefore, finding the right treatment for the treatment of A. baumannii infections.

The inefficiency of synthetic antibiotics against drug-resistant bacteria has led to a re-emergence of interest in silver, which has a long history as an antimicrobial agent. 10 – 12 Antimicrobial activity of silver nanoparticles (AgNPs) has been reported against many species of bacteria; for example, Escherichia coli ATCC 8739, 13 Staphylococcus aureus ATCC1431, 14 Escherichia fergusonii, and Klebsiella aerogenes ATCC 1950, 15 among others. AgNP synthesized with protective agents, such as citrate, sodium dodecyl sulfate and polyvinylpyrolidone showed increased antibacterial activity against S. aureus and E. coli. 16 Huang et al. Reported antimicrobial activity against A. baumannii with the synergistic combination of chitosan acetate and AgNPs. 17 Jain et al. Investigated the interaction of AgNPs with commonly used antibiotics in Pseudomonas aeruginosa, 15 while Morones-Ramirez et al. Demonstrated that Ag +-treated Gram-negative bacteria were susceptible to vancomycin. Gram-positive specific antibiotics, both in vitro and in vivo. 18 However, the synergistic antibacterial activity of the antibiotic in combination with citrate-containing silver nanoparticles has not been studied.

In this study, we investigated the synergistic effect of antibiotics with nano-silver against resistant A. baumannii bacteria obtained from clinical patients both in vivo and in vitro. We have also investigated possible mechanisms of this synergistic effect.

Materials and methods

Material

Trisodium citrate, silver nitrate (AgNO3) and sodium borohydride (NaBH4) are used to synthesize AgNPs. Rifampicin, tigecyline, polymyxin B (PMB), mucin, dimethyl sulfoxide, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), isopropyl-β- D-thiogalactoside, penicillin – streptomycin and trypsin – ethylenediaminetetraacetic acid purchased from Sigma Aldrich (St Louis, MO, USA). Cells A549 and cell HL-7702 were purchased from the Chinese Academy of Sciences’ Type Cultural Collection (Shanghai, People’s Republic of China). Roswell Park Memorial Institute-1640 medium was purchased from Thermo Fisher Scientific (Waltham, MA, USA) and fetal serum was purchased from HyClone (Logan, UT, USA). No ethical committee approval for this test set is required as the test was performed on commercially available cell lines and is considered to be a full evaluation waiver by the ethics committee at Jiaotong University. through Shanghai.

All animal procedures have been approved by the Foundation’s Animal Care and Use Commission at Shanghai Jiaotong University. All animal studies are performed in accordance with the Guidelines for the Care and Use of Laboratory Animals in accordance with the People’s Republic of China Regulations for Laboratory Animal Administration. All animal procedures were approved by the Animal Ethics Committee of Shanghai Jiao Tong University. Mouse C57BL / 6 was purchased from Slac Laboratory Animal Co., Ltd. (Shanghai, People’s Republic of China).

Synthesize and describe characteristics of AgNPs

In a flask with a three-neck round bottom, 20 ml of trinatri citrate (1%) and 75 mL of ultra-pure water were mixed for 15 minutes at 70 ° C. Add 1.5 mL of silver nitrate (1%) solution. ); NaBH 4 (1%) was then added, then quickly mixed. This mixed solution is heated for 60 minutes, cooled to room temperature and made up to 100 mL with water.

To describe the morphology of the AgNP synthesized, the transmission electron microscope (TEM) analysis was performed by using TEM Tecnai G2 Spirit 120 kV device (resolution 0.23 nm) (FEI Company , Hillsboro, OR, USA).

AgNP is also characterized by scanning absorption spectra in the 300–500 nm range with a multifunctional full microwave analyzer (BioTek Co., Winooski, VT, USA).

A Malvern Zetasizer Nano-ZS device (Malvern, Louis, USA) was used to describe the zeta potential of nanoparticles in solution. Data was collected and analyzed using Zetasizer software (Malvern, Louis, USA).

Determine the minimum inhibitory concentration and the fractional inhibitory concentration

Bacterial strain A. baumannii (aba 1604; Huashan Hospital Phuc Dan University, Shanghai, People’s Republic of China) was used as a model test strain to determine the antibacterial activity of nano-silver. Different concentrations of AgNP were incubated with bacteria 4 × 105 in Luria Bertani broth (LB) in 96-well round bottom plates. The bacteria were harvested at the specified times and the optical densities of the samples tested at 600 nm. All samples were plated three times and values ​​were averaged from three independent tests. A. baumannii resistance (aba1604) was created from clinical patients and followed institutional ethics guidelines that were reviewed and approved by the ethics committee at the Huashan Hospital clinical ethics committee, learn Fudan. Shanghai Laiya R & D Biopharmaceutical Center, which has collected these strains for antimicrobial research and collected these samples is not necessary as sample collection is part of disease care. regular multiplication.

To evaluate the antibacterial activity of nano-silver in combination with antibiotics, a two-dimensional microscopic dilution assay was used. 19 The test was performed in LB culture media. The first estimate of the minimum inhibitory concentration (MIC) for each antibiotic and the fractional inhibitory concentration (FIC) of the combination of the antibiotic and AgNP was then determined by a bench microscopic method. flags in the 96-well microplate. Antibiotics and nano silver are diluted to the following concentrations (2MIC, 1MIC, 1 / 2MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC and 1/32 MIC) in the micro-dilution test. two-way.

The plates were incubated at 37 ° C for 18 hours, and the results were checked by measuring optical density (OD) 600. The combined antibiotic effect of agents A and B (where A is AgNO 3 or AgNPs and B is one of the three antibiotic agents) is calculated as follows:

FIC index values ​​above 4.0 indicate antagonistic effects, values ​​between 0.5 and 4.0 indicate additive effects, and values ​​less than 0.5 indicate effects synergistic

Cell toxicity test

To determine the cytotoxic activity of silver nanoparticles on mammalian cells, cells A549 and cells HL-7702 (1 × 10 4 cells / mL) were cultured in Roswell Park Memorial Institute-1640 medium. Contains 5% fetal serum in a 96-well plate. at 37 ° C in an environment with 5% CO 2 for 24 hours. Cells were treated with AgNPs, AgNO 3, or reference solution at a concentration of 0.625 to 10 μg / mL for another 24 hours. To determine viability, MTT (at a concentration of 0.1 mg / mL) was added to the well and incubated for 4 hours at 37 ° C and 5% CO 2 to allow cell growth. 21 In the metabolically active cells, MTT is reduced to insoluble, dark purple formazan. The purple formazane is then dissolved in dimetyl sulfoxide. Absorbance was measured at a wavelength of 570 nm using a multifunctional full-wavelength microfiber analyzer, and the readings were compared from untreated cells. The OD values ​​are used to sort the possible percentage of cells by using the following formula:

Tested for antimicrobial activity in vivo

Minimal lethal dose of AgNO 3 or nano-silver in rats

Six-week-old C57BL / 6 male mice (body weight ~ 20 g) were used for all animal experiments. Rats are kept in a controlled environment of temperature and humidity, and have free access to food and drinking water. Ten rats per group were injected into the peritoneum with a total volume of 100 μL. Rats were treated as follows: no treatment, and 10, 20, 40 and 80 mg / kg AgNPs and AgNO3. The injected animals were observed for 3 days.

Determination of minimum lethal dose of A. baumannii in a rat model of peritonitis

Dilutions of serial A. baumannii 1 × 107 to 1 × 1011 CFU, in 500 μL of sterile saline with the addition of 8% mucin, were injected into the peritoneum of the rat. 18 animals were observed for 2 days to determine survival.

Survival test

The mice were injected into the peritoneum with the minimum lethal dose (MLD) of A. baumannii, with a total volume of 500 μL with 8% mucin. After 1 hour, ten rats in each group were injected into the peritoneum 100 μL of vehicle phosphate buffer solution (PBS) or one of the various antimicrobial treatments. Rats were observed for 2 days to assess survival.

Tests for bacterial penetration

The surviving rats were operated on and cut down, and their kidneys and lungs were collected. These organs are ground under aseptic conditions, and the homogeneous substances are dissolved in disinfectant saline. These visceral homogeneities were then cultured on LB plates at 37 ° C for 24 hours.

The Cytokine Profile

The cytokine concentration in rat plasma was measured at the indicated time after infection with a standard enzyme-linked immunosorption test kit according to the manufacturer’s instructions (Elabscience Biotechnology Co., Ltd, Vu. Han, People’s Republic of China).

Antisense RNA model to detect synergism of AgNP and antibiotic combination

To investigate the pathways involved in bacterial responses to silver nanoparticles, we conducted a series of experiments, which examined the effects of AgNPs and AgNO3 on different bacterial gene silencing strains. E. coli induces RNA. 22 – 24 Different silenced gene strains were arranged in microwave plates, and then screened to determine their sensitivity to each different Ag formula compared to the parental strain. Genetically reduced E. coli strains were treated with isopropyl-β- D -thiogalactoside at the appropriate concentration (Table 1), and cultured fractions were transferred to a 96-well plate. Dilute concentrations of nano silver, AgNO3, rifampicin, tigecyline, and PMB were added to the gene silencing bacteria in 96-well plates. Finally, plates were incubated at 37 ° C for 16 hours and shaken at 80 rpm. The absorbance is measured at 600 nm with a fully functional full wavelength microfiber analyzer, and the OD values ​​are used to calculate the inhibition rate (I) using the following equation:

Statistical analysis

Each test was repeated three times. Data are presented as mean ± standard deviation, unless otherwise noted. Comparisons between multiple groups were performed using one-way analysis of variance (ANOVA) and all analyzes were performed using statistical software SPSS 21.0 (21.0; IBM Corporation, Armonk, NY, USA). The threshold of statistical significance is set at P <0.05.

Result

The properties of nano silver

Based on the image in Figure 1A, the AgNPs stabilized by citrate have good dispersion. The particle size distribution was shown by counting the number of AgNPs based on the TEM image and the particle size distribution plot in Figure 1B. AgNPs are 5-12 nm in diameter, with an average size of 8.4 nm. The nanoparticles were stable for more than 6 months, even at 37 ° C. The zeta potential of the AgNP synthesized is summarized in Figure 1C. Visible ultraviolet (UV – vis) spectra of solution samples are reported in Figure 1D. A single strong peak was observed at a wavelength of 392 nm, indicating the synthesis of spherical nanoparticles. In practice, the dispersion is stable if the zeta potential is higher than 30 mV or less than −30 mV. In support of the earlier claim, we observed that the AgNPs dispersed in water were very stable with a zeta potential value of -44.5 mV.

Figure 1 Appearance and physical and chemical properties of AgNPs. Note: (A) TEM of AgNPs. (B) Size distribution of AgNPs based on TEM image. (C) Analysis of the Zeta potential of AgNPs. (D) Visible UV absorption spectrum showing maximum absorbance at 392 nm for AgNP.

Antibacterial activity of nano-silver combination treatments

Combined antibiotic therapy is a strategy commonly used in the treatment of multidrug-resistant A. baumannii (MDR). Because PMB, rifampicin, and tigecycline are all commonly used against MDR A. baumannii in combination with other antibiotics, 25-27, we chose three antibiotics to evaluate potential synergistic effects. with nano silver. AgNPs were found to exhibit strong antimicrobial activity against A. baumannii, with an MIC of about 2.5 μg / mL, similar to that of AgNO3. The MIC for the typical drug, summarized in Table 2, is 0.25 μg / mL for PMB, 3.12 μg / mL for rifampicin and 3.12 μg / mL for tigecycline.

Table 2 FIC index of silver and antibiotic combination against Acinetobacter baumannii. Note: Data are presented as average ± SD, unless otherwise stated.

The FICs of AgNPs and various antibiotic combinations have been studied and are summarized in Table 2. These experiments showed that PMB and rifampicin were synergistic (P <0.5) with AgNPs and AgNO3, while tigecycline did not show synergies (P> 0.5) with AgNPs or AgNO3.

Cell toxicity of nano silver in vitro

Several investigations have reported inhibitory effects of AgNPs on cells. For example, Beer et al. Found that AgNPs inhibited proliferation of A549 cells in a dose-dependent manner, 28 while Foldbjerg et al. Reported that AgNPs caused an increase in reactive oxygen levels ( ROS) in cell A549. 29 Here, we use the method described by Foldbjerg et al. To evaluate the cytotoxicity of AgNPs. 29

As shown in Figure 2, a high concentration of AgNO 3 significantly affects cell growth. For comparison, exposure to AgNPs at concentrations higher than 10 μg / mL showed no significant cytotoxicity in cells A549 and HL-7702. These results demonstrate that the cytotoxicity of AgNPs is lower than that of AgNO3

Figure 2 Relative survival rate of cells A549 and HL-7720 exposed to AgNPs. Note: Relative survival of cells is influenced by different doses of AgNPs or AgNO3. (A) MTT test results confirm in vitro cytotoxicity of AgNPs and AgNO 3 against cells A549. (B) Effect of AgNPs or AgNO 3 on HL-7720 cell growth. Results are expressed as the mean ± SD of the three independent experiments. * P <0.05, ** P <0.01 and *** P <0.001 compared to the control.

Effect of nano-silver on in vivo antibacterial activity

Acute toxicity of AgNPs and AgNO 3 was measured in vivo to establish a mean lethal dose (LD 50) (Figure 3). The LD 50 for AgNPs was defined as 20 to 40 mg / kg, and the LD 50 for AgNO 3 was also found to be between 20 and 40 mg / kg. These LD 50 values ​​for AgNO 3 are similar to those reported in a previous toxicity study.

Figure 3 Toxicity of AgNPs and AgNO 3 in mice. Notes: (A) Survival rates of rats receiving the following treatments: control, 10, 20, 40 and 80 mg / kg AgNO 3. (B) Survival rates of rats receiving the following treatments: control, 10, 20, 40, or 80 mg / kg AgNPs. Survival trials were performed with ten rats per group.

Infections were formed in rats through distribution of 5 × 10 9 A. baumannii cells in the peritoneum suspended in an aqueous solution containing 8% mucin. Within 24 hours of injection, all infected mice died. Therefore, A. baumannii was used at this same concentration of 5 × 10 9 cells for all subsequent experiments.

The combination of PMB and AgNPs and the combination of PMB and AgNO 3 showed the same synergistic antibiotic effect that we had previously observed in vivo (Figure 4). We observed survival of rats for 1 week, and calculated 2-day survival. A mixture of AgNO3 and PMB (3 mg / kg and 10 μg / kg respectively) resulted in 40% survival. When either of these compounds is used alone as a single-dose treatment, the survival rate is 0%. For comparison, even a low dose mixture of AgNPs and PMB (2 mg / kg and 10 μg / kg respectively) resulted in a high 60% survival rate.

Figure 4 Survival rates of mice injected with AgNPs or AgNO 3 with PMB in a peritonitis infection pattern. Notes: (A) PMB therapeutic concentration in peritonitis infection model. (B) AgNO 3 and PMB therapeutic concentrations in peritonitis infection model. (C) AgNPs and PMB therapeutic concentration peritonitis infection model.

Two days after using the drug in mice, we cut a part of the mouse to test for the penetration of the bacteria. A representative picture of bacterial growth is shown in Figure 5, which shows that AgNO3 and AgNPs enhanced PMB activity against A. baumannii in the peritonitis infection model. Figures 5A and B show that there were a lot of bacteria present in both the kidneys and lungs when animals were treated with PMB alone at a dose of 250 μg / kg. With the addition of AgNO 3 (3 mg / kg, 18 μM) or AgNPs (2 mg / kg, 18 μM), no bacteria were detected in the kidneys or lungs. When we reduced the PMB dose to 50 μg / kg, we found that the kidney and lung tissues contained only small amounts of bacteria. The further reduction of the PMB dose to 10 μg / kg is less effective, leaving a significant bacterial burden in the kidneys and lungs. Furthermore, we tested the blood and ascites of infected rats and found that they did not contain bacteria when using a combination of PMB (50 μg / kg) with AgNO 3 (3 mg / kg). , 18 μM) or AgNPs (2 mg / kg, 18 μM) (Figures 5C and D). These results not only show that AgNO 3 can enhance the antibacterial activity of the antibiotic, but AgNPs are also antimicrobial in vivo.

Figure 5 Bacterial burden in mice infected with Acinetobacter baumannii after treatment with PMB in combination with AgNO3 or AgNPs. Note: bacterial burden in mice infected with Acinetobacter baumannii after treatment with PMB (250 μg / kg) (a); mice were infected after treatment with PMB in combination with AgNO 3 (3 mg / kg, 18 μM) (b1: 250 μg / kg; b2: 50 μg / kg; b3: 10 μg / kg), and with AgNPs (2 mg / kg, 18 μM) (c1 – c3). (A) kidneys; (B) lungs; (C) blood; (D) ascitic liquid.

To analyze whether AgNO3 or nano silver in combination with antibiotics modulates inflammation during A. baumannii infection, we evaluated proinflammatory cytokines in rat plasma by an immunosorption assay. binds to enzymes. Tumor necrosis factor levels alpha (TNF-α) and interleukin (IL) -6 were significantly reduced in the rat plasma of the treated mice compared with the model mice at 18 hours post-infection ( Figure 6). AgNO 3 and AgNPs may enhance PMB activity against A. baumannii in vivo.

Figure 6 AgNO 3 or AgNPs associated with inflammatory response by Acinetobacter baumannii prepared by antibiotics.
Note: ELISA was used to measure IL-6 (A) and TNF-α (B) in mouse plasma 18 hours after infection. Results are expressed as the mean ± SD of the three independent experiments. *** P <0.001 compared to

Mechanism of enhancing antibacterial activity of nano silver in combination with antibiotics

To better understand the mechanism supporting the synergistic action of nano silver in combination with antibiotics, we used the gene silencing function caused by the reactive RNA to suppress the expression of some genes in E. coli (Figure 7). Differences in inhibition rates between genetically silent E. coli strains and control E. coli strains DH5α / pHN678 were color-represented in our heat mapping analysis; The dark red color represents the most susceptible strains, while the dark blue color represents the least susceptible strains, compared to the parent strain. We found that silencing of rpoD, kdsA, kdsB, lpxC and mutL resulted in sensitivity to both AgNO 3 and AgNPs. Silence of psR, rpsL, rpsA, murB, murA, leuS and dnaB only leads to sensitivity to AgNO 3, which indicates that silver nanoparticles operate at higher selectivity than AgNO3.

Figure 7 Analysis of the temperature map of susceptibility to AgNPs and AgNO 3 in the genetically reduced Escherichia coli strains compared with the control E. coli DH5α / pHN678.
Note: Each unit exhibited a difference in inhibition rates between a single gene silencing antagonistic RNA and control E. coli DH5α / pHN678. The red units represent the strains most sensitive to compounds and the blue color represents the strains that grow similar to the E. coli control strain DH5α / pHN678.

In addition, the rpoD-resistant strain was susceptible to rifampicin, while the silence of kdsA, kdsB and lpxC increased the sensitivity to PMB, 42 increased the sensitivity of E. coli to AgNPs and AgNO 3. Most of the factors investigated were in the σ70 family, and all bacteria expressed one or more factor σ70. Factor σ70 sequence is highly conserved and plays an important role in bacterial growth. Rifampicin has a molecular mechanism of action involved in the inhibition of DNA-dependent RNA polymerase. 43 In E. coli, this enzyme is a complex oligomer consisting of four subunits: α, β, β ′ and σ, encoded by rpoA, rpoB, rpoC, and rpoD, respectively, and their disruption. interfere with transcription. 43 Potential mechanisms of both rifampicin and AgNP are involved in their effects on DNA-dependent RNA polymerase, which is evidence supporting their synergistic effects (Figure 8).

Figure 8 Proposed mechanisms for the combination of AgNPs / Ag + with antibiotics against G + negative bacteria.
Note: AgNPs / Ag + may enhance the damage caused by PMB to membrane lipids. AgNPs / Ag + and RIF can also bind to intracellular proteins and RNA polymerase when entering cells.

Silver and silver-containing compounds have recently attracted increasing interest as antimicrobial agents for the treatment of bacterial infections.Nano silver shows the synergy of biofilm inhibition. P. aeruginosa when combined with the lower MIC level of aztreonam. 44 The association of AgNP with ceftazidime has also shown synergy to inhibit P. aeruginosa. 45 AgNP prepared as described by Tiwari et al. Demonstrated strong antibacterial activity against carbapenem-resistant A. baumannii strain. 3 This requires an effective treatment regimen and the combination of rifampin with imipenem has been evaluated in clinical infections due to imipenem-resistant A. baumannii. Stress, overload. 46 Yoon et al. Showed that the association of PMB with imipenem was as effective as PMB with rifampin against a carbapenem-resistant A. baumannii strain. 26 Consequently, it is becoming increasingly important to drug therapy with newer antibiotics or a combination of antibacterial agents to eradicate these infections. According to our research, the combination of AgNP with antibiotics could be an effective solution to the problem of carbapenem-resistant A. baumannii strains, which are likely at lower doses and less toxic than currently available. This is often used clinically.

Previous investigators have suggested that combination drug treatment could be an effective tool to prevent the emergence of drug-resistant bacteria, especially in patients infected with Gram-negative bacteria. developed resistance to a single therapy. 47 Our study showed a synergistic effect of combining AgNPs and PMB or AgNPs and rifampicin against drug-resistant A. baumannii isolated from clinical patients. Considering the lower toxicity of AgNPs compared to other treatment options, these drug combinations have the potential to be useful tools for the clinic.

Reference source: Effects of silver nanoparticles in combination with antibiotics on the resistant bacteria Acinetobacter baumannii

Guoqing Wan,^1,2 Lingao Ruan,^2,3 Yu Yin,^2,3 Tian Yang,^2,3 Mei Ge,² Xiaodong Cheng^1,4

¹School of Life Science and Technology, China Pharmaceutical University, Nanjing, ²Shanghai Laiyi Center for Biopharmaceutical R&D, ³School of Pharmacy, Shanghai Jiao Tong University, Shanghai, People’s Republic of China; ⁴Department of Integrative Biology & Pharmacology, The University of Texas Health Science Center, Houston, TX, USA