*3.4. Patients*

The study protocol and the letter of informed consent were approved by the local Hospital's Ethics and Science Committee with the number 102-16.

The study included urine samples from patients with urinary catheters treated at the local Hospital. Patients were selected in basis to NOM-045-SSA2-2005. For the purposes of this study, only samples of patients older than 15 years, of any genus, whose culture had a bacterial count greater than or equal to 50,000 CFU/mL were selected, and also with an antimicrobial resistance profile.

#### *3.5. Microbiological Analysis*

#### 3.5.1. Sample Collection and Bacterial Culture

Urine samples were collected in 5 mL syringes using the probe puncture technique under aseptic conditions by trained personnel. Microbiological analysis was performed according to the established criteria by the American Society for Microbiology (ASM) for urine samples. A count greater than or equal to 50 colonies (equivalent to 50,000 CFU/mL) was considered as the cutoff point for diagnosing infection. Plaques without development at 24-h incubation were discarded and indicated absence of urinary tract infection.

#### 3.5.2. Identification and Antimicrobial Susceptibility Profile of Clinical Strains

The identification and antimicrobial susceptibility of the microorganisms isolated were determined by VITEK2 equipment. From a pure culture grown for 24 h, an inoculum was transferred to a test tube with 3 mL of solution sterile saline (0.45% to 0.5% NaCl aqueous solution, pH 4.5 to 7.0). Subsequently, the turbidity was adjusted to 0.50–0.63 units of the McFarland scale with the densitometer. The bacterial suspension was placed inside the cassette. The identification and susceptibility cards were placed in the nearby slot, inserting the transfer tube into the test tube with the corresponding suspension. The cassette was placed with the samples in the VITEK2 system. The resistance profiles are shown in Table S1.

#### 3.5.3. Conservation of Strains

The conservation of the strains was carried out by the freeze conservation method. Glycerol was used as the cryoprotective agent. The samples were stored at −20 ◦C.

#### *3.6. Antimicrobial Test*

The Minimum Inhibitory Concentration (MIC) was determined by broth microdilution assay in accordance with the procedures recommended by the Clinical and Laboratory Standards Institute (CLSI) [34]. MICs were determined by incubating the microorganisms in 96-well microplates for 24 h at 37 ◦C. Microorganisms were exposed to serial dilutions of the antimicrobial agen<sup>t</sup> (AgNPs, ampicillin and amikacin), and the end points were determined when no turbidity in the well was observed. The standardization of the method was made based on the criteria established by the CLSI. The strains used were *Staphylococcus aureus* ATCC 25923 and *Escherichia coli* ATCC 25922. The antibiotics used were oxacillin (64 μg/mL) and ceftazidime (32 μg/mL). The assay was performed in duplicate for four days. The results of the standardization of the MIC are shown in Table S2.

## *3.7. Checkerboard Synergy*

The MIC of each antimicrobial substance alone or in combination was determined by a broth microdilution method in accordance with CLSI standards. The assay was performed in 96-well microtiter plates, a two-fold dilution of the antibiotic was distributed into each well to obtain a varying concentration of 128, 64, 32, 16, 8, 4, 2, 1, 05, 0.25 and 0.125 μg/mL in the wells of the first row, while those of the AgNPs were similarly distributed among the first column (128 to 1 μg/mL). The AgNPs dilutions were started from the columns to the right and the antibiotic dilutions were started from the first row downwards. Thus, each of the wells held a unique combination of concentrations of AgNPs and the antibiotic. The broth microdilution plates were inoculated with each test microorganism to yield the appropriate density (10<sup>5</sup> CFU/mL) in 100 μL Mueller–Hinton broth and incubated at the optimum temperature and time of growth conditions (37 ◦C/24 h). The MIC was determined as the least dilution without any turbidity. The MICs of single antimicrobial A and B (MIC A and MICB) and in combination were determined. The ten multidrug resistant clinical strains were exposed to serial dilutions of the antimicrobial agents. Subsequently, we calculated the proportion: MICAntibiotic alone/MICAntibiotic in combination (fold-change) to describe the number of times that MIC decreased from an initial to final value.

#### *3.8. Cytotoxicity of AgNPs*

To evaluate the toxicity of AgNPs in combination with antibiotics, human fibroblasts (baby foreskin) were used. Cells were dispensed in 96-well microplates at a density of 5000 cells per well in DMEM supplemented with 1% fetal bovine serum (FBS) for 24 h at 37 ◦C. After 24 h the cells were incubated with the established concentrations of AgNPs and antibiotics for 24 h at 37 ◦C. Treatment was withdrawn and 100 μL MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (0.5 mg/mL) was added in DMEM without FBS for 4 h at 37 ◦C for the formation of formazan crystals. MTT was removed from the wells and 100 μL of DMSO was added to read absorbance in a Synergy H1 microplate reader with Gen 5 software (Biotek Instruments, Winooski, VT, USA) at a wavelength of 595 nm. As a positive control, cells were treated with hydrogen peroxide and as a negative control, they were only treated with medium. The assay was performed in triplicate.

## *3.9. Statistical Analysis*

Each assay was repeated three times. Data are presented as mean ± standard deviation (SD). The comparison between the effects of the two sources of variation was made using the two-way analysis of variance (ANOVA). The analysis was performed with the statistical software SPSS 23.0 (IBM, New York, NY, USA). A value of *p* < 0.05 was considered significant.
