**1. Introduction**

Urinary tract infections (UTIs) are defined as inflammatory processes related to the invasion and multiplication of microorganisms that occur at any level of the urinary tract, including urethral (urethritis), bladder (cystitis), ureters (ureteritis) and kidney infections (pyelonephritis) [1].

Approximately between 150 and 250 million cases of UTIs occur each year worldwide [2]. In 2011, more than 8 million cases were reported in the U.S. [1], of which 93,300 were acquired in intensive care

units [3]. Recently, the epidemiological bulletin of the Ministry of Health reported in 2016 a total of 4,023,432 cases of UTIs in Mexico, of which 76.68% were in women and only 23.31% in men.

Gram-negative intestinal bacteria are the most common etiology agents of UTIs, where uropathogenic *Escherichia coli* (UPEC) is the major microorganism isolate, which is a member of the Enterobacteriacea family [4]. Other commonly associated pathogens include *Klebsiella* sp. and *Proteus mirabilis*, both of which are characterized by their urease enzyme and Gram-positive bacteria such as *Staphylococcus saprophyticus* and *Enterococcus faecalis* [5].

Urinary Tract Infections Associated with Catheters (CAUTI) are one of the most frequent explanations of nosocomial infections [6]. Patients with urinary catheters show an increment of bacteriuria in relation to duration of catheterization [7], however, the most important factor is the biofilm formation along the catheter surface [7,8]. A biofilm is a resistance mechanism that consists in a self-organized community of microorganisms embedded in a matrix of extracellular polymeric substances synthesized by themselves [9]. Many bacterial species show growth in the form of biofilms, which gives them various advantages [10]. Some of the benefits are metabolic cooperation (nutrients) [11], horizontal gene transfer [12], protection against environmental stresses, lower susceptibility to antimicrobial agents [13,14] and prevention of host defense mechanisms (immune system) [15]. The most common organisms that contaminate the urinary catheter and develop biofilms are strains of *Escherichia coli*, *Pseudomonas aeruginosa*, *Enterococcus*, *Proteus mirabilis*, *Klebsiella pneumoniae* and coagulase-negative staphylococci [10,16].

The antibiotic resistance of bacteria is a global health problem that is continually expanding, and is recognized as a medical problem that increases morbidity and mortality rates, which implies length of hospital stays as well as cost and bad prognosis [17,18]. In fact, the speed at which bacteria are establishing resistance to current antibiotics is faster than the development of new molecules with antimicrobial features. Unfortunately, it is very difficult to identify new bacterial targets that can be used to develop new classes of antimicrobial agents that are safe and effective.

In this context, nanotechnology opens new possibilities, allowing new solutions with old resources. Nanoscale materials such as silver nanoparticles (AgNPs) have emerged as novel agents due to their unique physicochemical properties and remarkable antimicrobial activities that confer a grea<sup>t</sup> advantage for the development of alternative products against, for example, multi-drug resistant microorganisms [19,20]. Due to the above, it has been proposed to implement the use of AgNPs on different devices for medical use. One of the strategies is the modification of surfaces of the devices to inhibit the formation of bacterial biofilms [21].

Recently, several studies have indicated that AgNPs can enhance the effect of antibiotics against susceptible and resistant bacteria, [22] as a well as decrease bacterial adhesion in the early stages of biofilm formation. In 2016, Rajendran and et al., impregnated urinary catheters with antibiotics (amikacin and nitrofurantoin) and a synergistic combination of antibiotics and AgNPs (synthesized by a biological method) in order to evaluate antibiofilm activity. The authors reported that the synergistic combination showed a 90% inhibition of bacterial adhesion, whereas functionalization with antibiotics showed only 25% inhibition [23].

Some authors have reported that AgNPs have toxic effects on mammalian cells; for example, impairment of normal mitochondrial function, increased membrane permeability and generation of reactive oxygen species [24,25].

In this study, the synergistic activity of AgNPs was evaluated with conventional antibiotics against Gram-positive and Gram-negative multi-drug resistant isolates from clinical samples. Results presented here show that AgNPs, in combination with antibiotics, increase the antimicrobial effect in an additive or synergistic manner. Furthermore, MTT assays sugges<sup>t</sup> that at low concentrations the AgNPs and their combinations do not present cytotoxic effects in eukaryotic cells.

#### **2. Results and Discussion**

#### *2.1. Synthesis and Characterization of AgNPs*

TEM micrograph revealed that the AgNPs were of spherical and pseudospherical shapes (Figure 1). Based on the particle size distribution histogram evaluated from the corresponding TEM micrograph (n = 100), the mean (±SD) size of AgNPs 8.57 ± 1.17 nm was calculated. The mean size for the combination of AgNPs with ampicillin (AgNPs + AMP) was 4.01 ± 0.80 nm and for the combination of AgNPs with amikacin (AgNPs + AMK) was 6.03 ± 0.87 nm (Table 1).

The particle size distributions of AgNPs + AMP and AgNPs + AMK were also evaluated in Dulbecco's Modified Eagle Medium (DMEM).

**Figure 1.** Morphological characterization of silver nanoparticles and their combinations with antibiotics. Transmission electron micrographs showing the formation of spherical and pseudospherical nanoparticles. (**a**) AgNPs; (**a'**) AgNPs in DMEM; (**b**) AgNPs + AMP; (**b'**) AgNPs + AMP in DMEM; (**c**) AgNPs + AMK; (**c'**) AgNPs + AMK in DMEM. Insets: Particle size distribution histogram. DMEM: Dulbecco's Modified Eagle Medium. AMP: Ampicillin. AMK: Amikacin.

AgNPs synthesized in aqueous solution and their combinations with antibiotics were characterized by DLS (Table 1). The results of the dialyzed AgNPs showed a narrow size distribution with a hydrodynamic diameter of (±SD) 8.23 nm ± 0.91 nm and a zeta potential value of −40.80 mV ± 9.54 mV. The combination of AgNPs + AMP also showed a narrow size distribution with a hydrodynamic diameter of 4.69 nm ± 0.51 nm. This decrease in size was attributed to the fact that ampicillin favors the homogeneous dispersion of the nanoparticles and gives it a greater stability when it obtains a zeta potential value of −51.00 mV ± 20.20 mV. The results of combination of AgNPs + AMK showed a narrow distribution of sizes with a hydrodynamic diameter of 947.90 nm ± 65.30 nm and a value of −21.10 mV ± 4.63 mV for zeta potential. The increase

of size of the nanoparticles are attributed to the addition of amikacin, which favors agglomeration of the AgNPs and causes an increase in the zeta potential, which translates in less stable nanoparticles.

The hydrodynamic diameters and zeta potentials of AgNPs and their combinations were also evaluated in DMEM.

**Table 1.** Characterization of silver nanoparticles and their combinations with antibiotics by DLS and TEM.


Data are expressed as mean and standard deviation. DLS: Dynamic Light Scattering. TEM: Transmission Electron Microscopy. DMEM: Dulbecco's Modified Eagle Medium. AMP: Ampicillin. AMK: Amikacin.

The UV–visible spectrum revealed a peak at a wavelength of 412 nm for AgNPs whereas in the combination AgNPs + AMP, the peak was observed at 410 nm and in the combination AgNPs + AMK, the peak appeared at a wavelength of 450 nm (Figure 2). These peaks correspond to the excitation of the surface plasmon of AgNPs. The plasmonic resonance depends on several parameters, like the nature, size and geometry of the nanoparticles and the physical properties of the medium in which the nanoparticles are dispersed. In the case of AgNPs, the plasmon peak appears at a wavelength around 400 nm [26].

**Figure 2.** UV–visible absorption spectra of silver nanoparticles and their combinations with antibiotics. UV–visible spectrum showed the maximum absorbance at (**a**) 412 nm for AgNPs, (**b**) 410 nm for AgNPs + AMP and (**c**) 450 nm for AgNPs + AMK. AMP: Ampicillin. AMK: Amikacin.

In this study, gallic acid was used as a reducing and stabilizing agent, a molecule with a carboxylic group and rich in hydroxyl functional groups. Yoosaf et al. proposed that AgNPs are stabilized by gallic acid through electrostatic interactions through their oxidized carboxylic group and the afore-cited hydroxyl groups were capable of forming hydrogen bonds [27].

To study the possible interactions between antibiotics and the surface of the AgNPs, FTIR was performed (Figure 3). FTIR is useful for determining the chemical composition of antibiotics involved in the coating of AgNPs. The observed intense bands were compared with standard values to identify the functional groups. The FTIR spectra of the antibiotics (Figure 3a,b) changed greatly upon the combination with AgNPs (AgNPs + AMP and AgNPs + AMK), as displayed in Figure 3a',b'. Ampicillin is a molecule that has carbonyl and amine functional groups, while amikacin is a molecule rich in hydroxyl and amine groups. In the case of ampicillin, the band at 1759 cm<sup>−</sup><sup>1</sup> disappeared completely (Figure 3a'), which suggests that the antibiotic interacts with the AgNPs through its carbonyl group (C=O). Furthermore, the peak of the primary amine at 3380 cm<sup>−</sup><sup>1</sup> (Figure 3a) shifted to 3130 cm<sup>−</sup><sup>1</sup> (Figure 3a') after the combination with nanoparticles, indicating that the amine functional group was involved in the interaction with the surface of the AgNPs. Therefore, the spectrum of the amikacin showed that the bands at 1626 cm<sup>−</sup><sup>1</sup> corresponding to a carbonyl group and 3400 cm<sup>−</sup><sup>1</sup> for a primary amine disappeared completely (Figure 3b') after being combined with the nanoparticles. Those results of the FTIR sugges<sup>t</sup> that the functional groups of the antibiotics could be involved in the interaction by hydrogen bonds with gallic acid [28,29]. The results showed in Figure 3 are in concordance with the previous results of the Hua Deng et al., 2016, who carried out UV–Vis and Raman spectroscopy studies reveal that amikacin can form complexes with AgNPs, while ampicillin do not [30]. The authors reported that no Raman enhancement is observed when AgNPs are combined with ampicillin at any test concentrations. This implies that the antibiotics do not strongly interact with AgNPs to replace the stabilizer molecules on the surface of AgNPs. Moreover, they infer that the combinations of antibiotics and AgNPs have different ways to develop antimicrobial activities.

**Figure 3.** FTIR spectra of the antibiotics and their combinations with silver nanoparticles. (**a**) AMP: Ampicillin; (**a'**) AgNPs + AMP; (**b**) AMK: Amikacin; (**b'**) AgNPs + AMK. Insets: AMP and AMK structure.

#### *2.2. Samples and Bacterial Strains*

The multidrug resistance clinical strains used for this experiment were isolated from the urine of patients with CAUTI; microbiological analysis showed that the clinical pathogenic strains isolated were in accordance with the main etiologic agents causing CAUTI; these results are in accordance with previously results reported when the UTI were evaluated on hospitalized patients in Kolkata, an eastern region of India, as well as in studies where complicate and non-complicate UTI were studied [4,31].

#### *2.3. Antimicrobial Test*

A set of ten clinical pathogenic strains resistant to antibiotics associated with CAUTI were evaluated, of which two corresponded to Gram-positive strains and the rest to Gram-negative strains. The results showed that all clinical isolates (*E. faecium*, *S. aureus*, *A. baumannii*, *E. cloacae*, three different isolates of *E. coli*, *K. pneumoniae*, *M. morgannii* and *P. aeruginosa*) showed a MIC to AgNPs between 4 and 16 μg/mL. The bacterial strains showed MIC values of 4–128 μg/mL for amikacin and all Gram-negative strains were resistant to ampicillin.

#### *2.4. Checkerboard Synergy*

Table 2 shows the MIC archived with the ten multidrug resistance clinical strains grown in Mueller Hinton Broth with amikacin and ampicillin, both in the absence of AgNPs and when present; when the

bacteria were incubated with the combination of AgNPs and antibiotic (AgNPs + AMK and AgNPs + AMP), the ampicillin and amikacin MICs decreased drastically for all strains. The combination of AgNPs + AMK reduce the MIC by 2 to 32-fold. By contrast, with the combination of AgNPs + AMP reduce the MIC just with *S. aureus* and *E. cloacae* by 1- and 4-fold respectively; with the other microorganisms, the MICs were reduced by 16- and 32-fold. These results show a better antimicrobial activity of the combination of AgNPs + AMP, which could be explained by the interaction between the AgNPs and ampicillin and, therefore, arrangemen<sup>t</sup> of the molecules in a new compound, which could work in both ways, like independent chemical entities or a new compound; more experiments are needed to explain the role and proportion of each one of them.


**Table 2.** Efficacy of silver nanoparticles, antibiotics and their combinations against clinical strains.

\* Minimum Inhibitory Concentration (MIC) represents the concentration of antibiotic (amikacin or ampicillin) present in the combination. AMK: Amikacin. AMP: Ampicillin. \*\* The numbers in parentheses indicate that *E. coli* corresponds to a different clinical sample.

The synergistic effects of AgNPs and conventional antibiotics were evaluated by determination of the Fractional Inhibitory Concentration (FIC) index (Figure 4). Synergistic interactions of AgNPs and amikacin were observed against *Acinetobacter baumannii*, *Escherichia coli* (508) and *E. coli* (ATCC 25922). Synergistic interactions of AgNPs and ampicillin were found only against *Acinetobacter baumannii*. Other combinational activities of AgNPs and antibiotics were considered as partially synergistic interactions. These synergistic activities of AgNPs in combination with conventional antibiotics sugges<sup>t</sup> that it may be possible to reduce the viability of bacterial strains at lower antibiotic concentrations (Table 3).

**Figure 4.** Example of checkerboard testing. Blue circles denote the MIC of antimicrobial agents (alone) and blue line denote the FIC (combination of both). MIC: Minimum Inhibitory Concentration. FIC: Fractional Inhibitory Concentration.


**Table 3.** FIC index of combinations among silver nanoparticles and antibiotics against clinical and reference strains.

FIC: Fractional Inhibitory Concentration. AMK: Amikacin. AMP: Ampicillin. ATCC: American Type Culture Collection. The FIC index was interpreted as follows: FIC ≤ 0.5, Synergy (S); 0.5 ≤ FIC < 1, Partial Synergy (PS); FIC = 1, Additive (AD); 2 ≤ FIC < 4, Indifferent (I); FIC > 4, Antagonism (AN) [32,33]. \*\* The numbers in parentheses indicate that *E. coli* corresponds to a different clinical sample.

#### *2.5. Cytotoxicity of AgNPs*

The cytotoxicity of AgNPs and antibiotics was evaluated separately and in combination by the MTT assay. AgNPs were tested at concentrations of 0.25, 1, 4, 16, 64 and 128 μg/mL in human fibroblasts. The percentages of living and dead cells were determined after 24 h of being exposed in contact with the AgNPs. AgNPs concentrations less than 4 μg/mL showed a cytotoxic effect that resulted in a death rate of 13.8% or less. However, concentrations greater than 64 μg/mL caused significant cell death of approximately 67%. In addition, antibiotics (ampicillin and amikacin) were tested at concentrations of 64, 32, 8, 2, 0.5 and 0.125 μg/mL in human fibroblasts. It was found that the viability percentage for each of the concentrations of ampicillin was greater than 80% and for amikacin greater than 76%. To evaluate the cytotoxic effects of the combination of AgNPs and conventional antibiotics, ten combinations of different concentrations (AgNPs μg/mL + antibiotic μg/mL: 128 + 64; 64 + 32; 32 + 16; etc.) were tested. It was found that there is no statistically significant difference between the two treatments (AgNPs + AMP and AgNPs + AMK) with respect to cell viability when the two-way ANOVA was performed (*p* < 0.05). However, combinations of 128 μgAgNPs/mL + 64 μg of antibiotic/mL and 64 μgAgNPs/mL + 32 μg of antibiotic/mL caused a statistically significant decrease in cell viability when compared with the rest of the combinations tested (*p* < 0.05) evidenced by the reduction of the mitochondrial activity. On the other hand, it is important to highlight that when AgNPs were combined with antibiotics, at concentrations equal to or less than 8 μg AgNPs/mL showed a viability percentage between 90–95% (Figure 5).

**Figure 5.** Viability of cells treated with combinations of silver nanoparticles and antibiotics. To measure cytotoxicity, fibroblasts were treated with increasing concentrations of AgNPs + AMP (red) or AgNPs + AMK (blue) (n = 3). Twenty-four hours after of addition of treatment cell viability was determined using MTT. Results are expressed as mean and standard deviation. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 by two-way ANOVA. Control: DMEM. AB: Antibiotic = Amikacin (AMK) or Ampicillin (AMP).

Interesting results were archived when two specific multi-resistant strains, *E. faecium* with resistance to vancomycin and *A. baumannii* with resistance to meropenem, were tested and both showed, with the combination of AgNPs + AMK, a reduction of the MIC by 32-fold, as well as with the combination of AgNPs + AMP of the MIC by 16-fold. In both cases, the cytotoxicity to fibroblasts, of the concentrations of AgNPs + Antibiotic which showed a reduction of MIC, showed a reduction with statistical significance (Table 4).

**Table 4.** Viability of fibroblasts treated with silver nanoparticles, antibiotics and their combinations using concentrations corresponding to the MIC value.


MIC: Minimum Inhibitory Concentration. AMK: Amikacin. AMP: Ampicillin.

#### **3. Materials and Methods**

#### *3.1. Synthesis of AgNPs*

For the synthesis of nanoparticles, AgNO3 (0.01 M) was used as a metallic precursor and gallic acid (0.1 g) was used as a reducer and stabilizer agent. NaOH (3 M) was used for pH regulation. AgNPs were synthesized by dissolving 0.0169 g of AgNO3 in 90 mL of deionized water and this solution was placed in a 250 mL reaction vessel. A total of 0.01 g gallic acid was dissolved in 10 mL of deionized water and was added to the AgNO3 solution with magnetic stirring. After the addition of gallic acid, the pH value of the solution was adjusted using a solution of NaOH 3 M. At the end of the synthesis, approximately 100 mL of nanoparticles were obtained with a pH of 12.66, of which 50 mL were dialyzed for 48 h on a 12 kDa nitrocellulose membrane.

#### *3.2. Characterization of AgNPs*

AgNPs were characterized by Dynamic Light Scattering (DLS), the hydrodynamic diameter and zeta potential were determined using a Malvern Zetasizer Nano ZS (Malvern Panalytical, Malvern, United Kingdom) operating with a He-Ne laser at a wavelength of 633 nm and a detection angle of 90◦. Samples were analyzed for 60 s at 25 ◦C. To confirm the shape, each sample was diluted with deionized water and 50 μL of each suspension was placed on a copper wire for Transmission Electron Microscopy (TEM). All samples were analyzed by Transmission Electron Microscopy (JEOL JEM-1230, Tokyo, Japan) at an acceleration voltage of 100 kV. Afterwards, AgNPs were characterized by UV-visible spectroscopy using an S2000 UV-Vis spectrophotometer from OceanOptics Inc. (Dunedin, FL, USA). The functional groups present in the antibiotics were identified by Fourier Transform Infrared Spectroscopy (Shimadzu, IRaffinity-1, Osaka, Japan). A certain amount of nanopowder was collocated in the equipment and the spectrum was taken in the range of 400–4000 cm<sup>−</sup><sup>1</sup> with a resolution of 2 cm<sup>−</sup><sup>1</sup> and 200 times scanning using the attenuated total reflection (ATR) method.

#### *3.3. Preparation and Characterization of Combinations of AgNPs with Antibiotics*

To study the effect of ampicillin and amikacin on the size, shape and stability of the AgNPs, an aqueous solution containing a 1:1 ratio of antibiotic (128 μg/mL) and nanoparticles (128 μg/mL) was prepared for each antibiotic. These solutions were characterized by TEM, DLS, zeta potential and UV-visible spectroscopy. On the other hand, the chemical interaction between the AgNPs and antibiotics was carried out by FTIR, we prepared an aqueous solution containing higher concentrations of antimicrobials (500 μg/mL), the combinations preserved the ratio of 1:1. Subsequently, these solutions were centrifuged, keeping only the precipitate, which was left to dry for 24 h at room temperature.
