An AH16 P. aeruginosa strain was isolated from a patient with chronic pneumonia in the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine (Hefei, China). This study was approved by the ethics committee of the First Affiliated hospital of Anhui University of Traditional Chinese Medicine. The strain was found to exhibit higher virulence and produce more biofilm when compared with the wild-type P. aeruginosa. SH and AZM were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Luria-Bertani (LB), Mueller-Hilton (MH) and Tryptic Soy Broth (TSB) media were purchased from Beijing Aoboxing Bio-Tech Co., Ltd. (Beijing, China), and crystal violet solution was purchased from bioMérieux, Inc. (Craponne, France). Alginate standards were purchased from Sigma-Aldrich (St. Louis, MO, USA), and Taq polymerase and PCR primers were purchased from Takara Bio, Inc. (Otsu, Japan).

The AH16 strain was inoculated into LB broth, and grown in a constant-temperature shaker (GLY; Fuma, Shanghai, China) at 220 rpm for 6 h at 37°C. Subsequently, the bacteria were harvested using a GL-20G-II high-speed refrigerated centrifuge (Fuma) at 1,630 × g for 10 min. The supernatant was discarded and the precipitate was resuspended with phosphate-buffered saline (PBS; pH 7.2), after which the samples were centrifuged again at 1,630 rpm for 10 min. The harvested cells were resuspended in PBS (pH 7.2) and adjusted to 2×10⁵ colony-forming units (CFU)/ml using the growth curve method (20).

Sauer et al (21) previously described five stages of biofilm formation as follows: i) Reversible attachment (0–2 h); ii) irreversible attachment (2 h); iii) maturation stage 1 (day 3); iv) maturation stage 2 (day 6); and v) dispersion (day 9–12). On the basis of these stages, days 1, 3 and 7 were selected as the three time points for observing the growth of the biofilm and to detect any suppression as a result of the drugs used. The minimum inhibitory concentrations (MICs) of SH and AZM were determined to be 512 and 16 µg/ml, respectively, using the microdilution method (22).

Fresh stock solutions of SH and AZM were prepared in MH broth and filtered through a 0.22-µm filter (EMD Millipore, Billerica, MA, USA). Six treatment groups were analyzed, which were treated with SH or AZM at a MIC of 0.5, 1 or 2 (Fig. 1). In a 96-well plate, 28 wells were filled with TSB medium, of which four wells were used for each of the six treatment groups, with four further wells used as the control. Next, 200 µl P. aeruginosa suspension was added and the plate was incubated at 37°C. After 24 h, the culture suspension was discarded, and the sedimented bacteria were washed in 1 ml PBS. Fresh TSB medium with the aforementioned concentrations of antibiotics was added to the relevant wells. The control wells contained TSB medium with no antibiotics. In accordance with the method previously described by O'Toole (23), the medium was exchanged for fresh medium with the appropriate antibiotics every other day. At the end of days 1 (attachment), 3 (maturation stage 1) and 7 (maturation stage 2), cold PBS (4°C) was used to wash the planktonic bacteria in one well of each group, after which 200 µl crystal violet solution (1%) was added to the wells and left for 20 min. The wells were rinsed with deionized water until no crystal violet was visible, following which the wells were dried and 95% alcohol was added for destaining. Subsequently, the destained solution from each well was transferred to a cuvette and diluted with 3 ml alcohol (95%). The optical density (OD) of the solutions was determined at 570 nm using a UV spectrophotometer (U-2000; Hitachi, Ltd., Tokyo, Japan). The OD value of the negative control was set as 100%, from which the growth of the biofilm was calculated for each group.

Alginate is the predominant constituent of the P. aeruginosa biofilm, and functions as a barrier to protect the bacteria from antibiotics and the humoral and cellular host defense system (24,25). Therefore, the effects of SH on alginate production in the P. aeruginosa biofilm were evaluated.

The groups analyzed were the same as those described previously (0.5x, 1x and 2x MIC groups each for SH and AZM). A sterile coverglass was used as a carrier for each group. The coverglasses were placed into the wells of a six-well culture plate, and 2 ml TSB medium and 0.2 ml bacterial suspension (2×10⁵ CFU/ml) were added. The plates were incubated for 1 day at 37°C, after which the medium was discarded. For each well, the coverglass was removed, and the planktonic bacteria on the coverglass were washed out with sterile PBS. The coverglass was subsequently returned to the well prior to the addition of fresh medium. As aforementioned, the medium added to the antibiotic groups contained the appropriate antibiotic, while the negative control contained medium without antibiotics. All the described steps were repeated every 24 h. At the end of days 1, 3 and 7 of the antibiotic treatment, the coverglasses were removed and the planktonic bacteria were rinsed with PBS. Subsequently, the coverglasses were each placed into a test-tube containing 6 ml PBS and 1.2 ml sulfuric acid and sodium borate compound which was prepared by dissolving 2.52 g sulfuric in 1:l sulfuric acid. The tubes were boiled for 5 min and then cooled to 4°C. Each coverglass received 20 µl hydroxybiphenyl (1%) for colorization, after which the coverglasses were sonicated for 30 min in an ultrasonic cleaning bath (2800; Branson Ultrasonics, Danbury, CT, USA) and the absorbance was measured at 570 nm using the Hitachi UV spectrometer (26). The alginate production of each strain, defined as the quantity of alginate (µg/ml) adhered to the coverglass, was calculated using a standard curve of alginate standards following the subtraction of the blank control.

Preparation of the carrier coverslips was performed as aforementioned, up to the point at which the planktonic bacteria were rinsed with PBS. Subsequently, the biofilm was subjected to silver staining (27), and cellular morphology was examined under SEM (Sirion 200 field-emission; FEI Company, Hillsboro, OR, USA) at 500 kV and x40,000 magnification.

RT-qPCR was performed to determine the expression levels of genes associated with alginate biosynthesis. The majority of the alginate biosynthesis genes are clustered in the algD operon (28). Alginate production is highly regulated, and AlgR is among one of the key regulators (29,30). Therefore, the algD and algR genes were selected for subjection to RT-qPCR in order to identify whether SH was able to attenuate the expression levels of alginate biosynthesis-associated genes. In addition, the expression levels of the biofilm-associated genes, pilL and rhlI, were evaluated.

Initially, 1 ml biofilm bacterial suspension culture, treated with 1x or 2x MIC concentrations of SH, was centrifuged at 10,800 × g for 1 min. The supernatant was discarded and the pellet was resuspended in 1 ml TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) at room temperature for 20 min. Each sample received 200 µl chloroform and was vortexed for 15 sec, followed by centrifugation at 4,200 × g at 4°C for 15 min. The liquid layer of the mixture was transferred to a fresh tube containing 480 µl isopropanol, after which the tube was vortexed for 15 sec and centrifuged at 4,200 × g at 4°C for 15 min. The total RNA was washed with 70% ethanol, and the tube was centrifuged again at 4,200 × g and 4°C for 10 min. The liquid was discarded, and the total RNA was dissolved in RNase-free water. Purified RNA (2 µg) was reverse transcribed into cDNA using a one-step method commercial kit (Takara Bio, Inc.). The primers were designed to produce products of 180–240 bp, and the sequences are detailed in Table I. The rpoD gene was used as a housekeeping control. PCR assays were conducted using an ABI Prism thermal cycler (Applied Biosystems Life Technologies, Foster City, CA, USA) using the following program: Initial denaturation for 5 min at 95°C, followed by 40 cycles of 95°C for 15 sec, 58°C for 10 sec and 72°C for 20 sec. In the experiment, there were three independent biological replicates and two technical replicates. The calculated threshold cycle (Ct) of each gene was normalized against the Ct of the rpoD gene amplified from the corresponding sample. Fold change was calculated according to the 2^−ΔΔCt method (31).

All data were analyzed by SPSS statistical software, version 17.0 (SPSS, Inc., Chicago, IL, USA), and expressed as the mean ± standard deviation. Difference between the various group data were compared using Students T-test. P-values were calculated by comparison of the data of drug treatment and control groups. Experiments were repeated in quadruplicate, and the results are expressed as the mean of the four replicates.

By day 1 of the antibiotic treatment, a significant effect on biofilm growth was observed in the 0.5x, 1x and 2x MIC SH treatment groups when compared with the control group (P<0.01; Fig. 1A), with the 1x and 2x MIC treatments producing a more marked effect compared with the 0.5x MIC treatment. This effect continued throughout day 3 (Fig. 1B) and day 7 (Fig. 1C), with the higher concentrations exhibiting the most notable effects. On day 7 (Fig. 1C), all the biofilms were reduced to <50% of the control (P<0.01). In addition, SH was more effective at repressing biofilm formation compared with AZM. These results indicated that SH significantly inhibited biofilm formation in P. aeruginosa (AH16) at various developmental stages.

At day 1 of the antibiotic treatment (Fig. 1D), there was a statistically significant (P<0.05) reduction in the alginate biofilm in the 2x MIC SH group when compared with the control group; however, this effect was not observed in the 0.5x or 1x MIC SH groups, which was similar to the effects of AZM. By day 3 (Fig. 1E), the 0.5x, 1x and 2x MIC SH treatment groups had induced significant reductions in alginate production (P<0.01) in a dose-dependent manner. At day 7 (Fig. 1F), the inhibitory effects of all three SH concentrations had produced reductions in the alginate production of ≥50% compared with the control. Notably, the inhibitory effects of SH were lower compared with AZM at 0.5x and 1x MIC, but were comparable to AZM at 2x MIC. These results demonstrated that SH exerts an inhibitory effect on alginate production in the P. aeruginosa AH16 clinical strain.

In the SEM images, the bacterial biofilm morphologies were observed to clearly vary between the two groups. In the control group (Fig. 2A), the bacteria were completely covered by a thick mucous biofilm, and the entire structure exhibited a mushroom shape with interlaced inner pore channels towards the surface. By contrast, in the 1x MIC SH treatment group (Fig. 2B), there was no evident biofilm structure and the rod-shaped bacteria were dispersed over the mucus. Thus, the morphology of the biofilm cells indicated the inhibition of alginate production by SH.

Expression levels of the algD and algR genes were reduced in the SH treatment groups when compared with the control group (Fig. 3). This reduction was dose-dependent, with the expression level being more notably reduced in the group treated with the highest concentration of SH (2x MIC) compared with the medium concentration (1x MIC). By contrast, the biofilm-associated genes, pilL and rhlI, were not downregulated transcriptionally by SH (data not shown). These results indicated that SH inhibits biofilm formation in P. aeruginosa by repressing the expression of genes associated with alginate biosynthesis.