The P. aeruginosa ATCC27853 strain was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The clinical isolate, PA012, was isolated from a patient with a urinary tract infection at and provided by the Clinical Laboratory of Weifang People's Hospital (Weifang, China). The present study was approved by the ethics committee of the Weifang People's Hospital. Written informed consent was obtained from the patient prior to the collection of PA012. The strains were stored at −80°C and subjected to cultivation in Luria-Bertani medium (Hope, Qingdao, China) with constant shaking at 8 × g at 37°C for 18 h until the strains reached the late logarithmic growth phase.

The minimum inhibitory concentrations (MICs) of BEB and IMP were assessed using the broth microdilution method following the criteria of the Clinical and Laboratory Standards Institute (no. M07-A10) (15). The bacterial suspension was adjusted to 1×10⁵ colony-forming units (CFU)/ml and incubated with BEB/IMP in a 96-well flat-bottomed microtiter plate for 24 h at 35°C. The concentrations of BEB/IMP were serially two-fold diluted in a range of 2–1,014 µg/ml for BEB and 0.25–128 µg/ml for IMP. The MIC was defined as the lowest concentration of BEB/IMP to cause no visual growth of bacteria. The combination of BEB and IMP was evaluated by checkerboard assay. The concentrations of BEB and IMP were set in a range of 1/64-1 MIC for BEB and 1/64–16 MIC for IMP. The fractional inhibitory concentration index (FICI) was determined using the following equation: FICI=(MIC_BEB in combination/MIC_BEB alone) + (MIC_IMP in combination/MIC_IMP alone), in which synergism, indifference and antagonism were interpreted as FICI ≤0.5, >0.5 and ≤4.0, and >4.0, respectively (16).

The initial PA012 suspension (1×10⁵ CFU/ml) was incubated with BEB and/or IMP at their concentrations at which synergism had been previously determined by using the checkerboard assay (17). The cells were counted at the designated time-points (0, 4, 8, 12 and 24 h). The control group remained untreated. T-K curves were plotted on a logarithmic scale of CFU/ml vs. time (h). Synergism was defined as an increase of ≥2log10 CFU/ml in the killing rate for the combined agents compared with the most active agent used alone. Antagonism was defined as a decrease of ≥2log10 CFU/ml in the killing rate for the combined agents compared with the most active agent alone. Indifference was defined as a decrease of ≤2log10 CFU/ml in the killing rate for the combined agents compared with any of the two agents used alone (17).

Prior to treatments with BEB (1/4 MIC) and/or IMP (1/8 MIC), a suspension of PA012 (100 µl; 1×10⁵ CFU/ml) was added into a 6-well flat-bottomed microtiter plate with sterile cover slips, followed by incubation for 24 h. The cover slips were then rinsed with sterile PBS, fixed with 2.5% (v/v) glutaraldehyde overnight at room temperature, and post-fixed with 0.1% osmium tetroxide for 1 h. The samples were subsequently dehydrated with a series of 30, 50, 70, 90 and 100% ethanol for 10 min each. After critical-point drying, the samples were processed by gold sputtering (JEOL JFC 1200E Ion sputtering device; JEOL Ltd., Tokyo, Japan) and observed by SEM (JSM-6700F; JEOL Ltd.).

PA012 suspension (100 µl; 1×10⁵ CFU/ml) was incubated with BEB (1/4 MIC) in combination with IMP at 1/16, 1/8, 1/4 and 1/2 MIC for 2 or 4 h. The adhesion test was performed on the surface of medical-grade polyvinyl chloride (Shengli Oilfield Central Hospital, Dongying, China). The medium was discarded and the wells were washed with pH 7.2 sterile PBS. The bacterial cells in each well were then stained with 150 µl 0.1% crystal violet for 15 min at room temperature. Following washing with PBS for several times and drying for 3 h, 150 µl 30% (v/v) acetic acid was added to the stained cells, followed by incubation with mixing for 15 min. The optical density at the wavelength of 492 nm (OD₄₉₂) was measured.

PA012 cells in the late logarithmic growth phase were centrifuged at 10,000 × g for 5 min at 35°C, washed twice with 0.01 M sterile PBS (pH 7.2) and resuspended in 1 ml PBS to reach an OD₆₀₀ of 0.2. The cells were then treated with BEB (1/4 MIC) or in combination with IMP at 1/16, 1/8, 1/4 and 1/2 of the MIC at 37°C for 10 min. Cells killed in a 100°C water bath were incubated with the same concentrations of agents were set as the control. Subsequently, the cells were centrifuged, washed and resuspended to reach an OD₆₀₀ of 0.15. The fluorescence of BEB was determined with a Synergy^™ HT multidetection microplate reader (BioTek, Winooski, VT, USA) at 360 nm excitation wavelength and 530 nm emission wavelength (18).

The PA012 cells (1×10⁵ CFU/ml) were first treated with BEB (1/4 MIC) and/or IMP (1/8 MIC). The total RNA was then extracted according to the instructions of the MagExtractor-RNA-kit (cat. no. NPK-200; Toyobo Life Science, Osaka, Japan). Complementary DNA was prepared following the instructions of the ReverTra Ace^® qPCR RT Master Mix with gDNA Remover kit (cat. no. FSQ-301; Toyobo Life Science). Primers with the following sequences were used: MexZ forward, 5′-CCCTTGTGAGGACGTTCAGT-3′ and reverse, 5′-CCAGCAACAGGTAGGGAGAA-3′; MexX forward, 5′-CATCAGCGAACGCGAGTACAC-3′ and reverse, 5′-CAATTCGCGATGCGGATTG-3′; MexY forward 5′-CCGCTACAACGGCTATCCCT-3′ and reverse, 5′-AGCGGGATCGACCAGCTTTC-3′; OprM forward 5′-GATCCCCGACTACCAGCGCCCCG-3′ and reverse, 5′-ATGCGGTACTGCGCCCGGAAGGC-3′; OprD forward 5′-ATCTACCGCACAAACGATGAG-3′ and reverse, 5′-GCCGAAGCCGATATAATCAAACG-3′; RpsL forward 5′-GCAAGCGCATGGTCGACAAGA-3′ and reverse, 5′-CGCTGTGCTCTTGCAGGTTGTGA-3′. The 25 µl qPCR mixture consisted of 12.5 µl 2X SYBR^® Green Real Time PCR Master mix, 1 µl PCR forward primer, 1 µl PCR reverse primer, 0.5 µl cDNA and 10 µl double distilled water. RT-qPCR was performed on an ABI7000 fluorescent qPCR system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The thermocycling steps were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 sec, 55°C for 10 sec and 72°C for 10 sec, and final elongation at 72°C for 5 min. All data were normalized to reference gene RpsL. The relative target-gene expression was calculated using the 2^−ΔΔCq method (19).

Results were obtained from three independent experiments performed in triplicate. Values are expressed as the mean ± standard deviation. Statistical analysis was performed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). Differences between groups were determined using one-way analysis of variance with a Student-Newman-Keuls method post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The MIC of BEB was 512 µg/ml in PA ATCC27853 as well as in PA012 cells, displaying weak antibacterial activity. Although the MIC of BEB in PA ATCC27853 cells was decreased 2-fold if used in combination with IMP at its MIC of 1 µg/ml, it was not synergistic with IMP, as the FICI was 1.5, which was interpreted as being indifferent. In the clinical isolate, PA012, the MIC of IMP was 265 µg/ml, and a synergism of BEB and IMP was identified with an FICI of 0.375 (<0.5). The MIC of BEB and IMP declined 4-and 8-fold, respectively, in combination compared with each alone (Table I). The synergism of BEB and IMP in PA012 cells was also evaluated by a T-K test. The results indicated that BEB (1/4 MIC) plus IMP (1/8 MIC) or IMP (1/8 MIC) alone significantly reduced the quantities of PA012 (P<0.05 vs. the control group), but the growth continued. The combination of BEB and IMP completely inhibited the bacterial growth, with a decrease of 3.4 log₁₀ CFU/ml compared with IMP, the most effective agent, alone (Fig. 1). Furthermore, the synergistic activity of BEB with IMP was confirmed by SEM evaluation, displaying a marked decrease in the density of PA012 cells compared with that in the control group (Fig. 2).

In the subsequent experiments, the concentration of BEB was set at 1/4 of the MIC to investigate the effects of its combination with a series of IMP at concentrations of 1/16, 1/8, 1/4 and 1/2 of the MIC on bacterial adhesion and BEB uptake into PA012 cells. It was observed that BEB (1/4 MIC) plus IMP (1/16 MIC) did not produce any evident inhibition of the bacterial adhesion at neither 2 nor 4 h, while application of BEB (1/4 MIC) plus IMP at 1/8, 1/4 and 1/2 of the MIC resulted in a marked decrease of bacterial adhesion at 2 h (P<0.05, P<0.05 and P<0.01, respectively) and at 4 h (P<0.05, P<0.01 and P<0.01, respectively; Fig. 3). BEB has fluorescent properties that may utilized for quantifying its intracellular accumulation. While significant fluorescence was detected in the BEB only group compared with the control, IMP significantly increased the fluorescence of BEB (P<0.01 or P<0.001; Fig. 4).

As demonstrated above, increasing concentrations of IMP promoted the intracellular accumulation of BEB in PA012. RT-qPCR was then used to analyze the antibacterial effects of BEB and IMP alone and in combination on the genes associated with the MexXY-OprM efflux pump in PA012. Compared with the control, BEB at 1/4 of the MIC and IMP at 1/8 of the MIC alone inhibited the expression of MexZ (P<0.05), MexX (P<0.05), MexY (P<0.01) and OprM (P<0.05, P<0.01). Of note, BEB plus IMP at the abovementioned concentrations resulted in decreases of 38% for MexZ (P<0.01), 35% for MexX (P<0.01), 46% for MexY (P<0.001) and 49% for OprM (P<0.001). However, OprD expression was not significantly affected by treatment with BEB and/or IMP (Fig. 5A). It was also demonstrated that the expression of OprD in PA012 was identical to that in PA ATCC27853, thereby excluding the possibility of OprD loss in response to the treatments (Fig. 5B).