Bacterial isolates
A total of 50 K. pneumoniae isolates were collected from the different departments of [Tanta University] Hospital. The clinical isolates were subjected to both microscopical examination and standard biochemical tests [10]. Klebsiella pneumoniae ATCC 13883 was used as a reference strain.
Chemicals
All the utilized chemicals in this study were of pure analytical grade and they were purchased from Sigma-Aldrich, USA.
Determination of MIC and MBC of BAC
The minimum inhibitory concentration (MIC) of BAC was identified by broth microdilution method in Muller-Hinton broth (MHB) (Oxoid, UK) in 96-well microtitration plates [11]. MIC is defined as the lowest antimicrobial concentration that inhibits bacterial growth indicated by the absence of turbidity in the wells compared to both the growth control and the uninoculated wells. The wells that showed inhibition of growth were subcultured on MH agar (MHA) (Oxoid, UK) for identification of the MBC (minimum bactericidal concentration) which is the lowest concentration of BAC that kills bacteria after overnight incubation at 37 °C. All these determinations were conducted in independent triplicates. As described by Moen et al. [12], bacterial isolates that show MIC > 12 μg/ml were estimated to be tolerant to BAC.
Adaptation to BAC
It was accomplished by daily exposing the tested isolates to sublethal concentrations of BAC with a gradual increase [13] beginning with a concentration of 0.5 × MBC of BAC. When the growth was detected, a 10-fold diluted culture was transferred to fresh MHB supplemented with a slightly higher concentration of BAC. This step was further continued until no growth was noticed after overnight incubation at 37 °C. Afterward, the bacterial suspension from the last tube that showed growth was inoculated to MHA and incubated overnight at 37 °C for growth conformation. All isolates were tested in triplicate.
Determination of the cell surface hydrophobicity (CSH)
It was identified before and after BAC adaptation as described by El-Banna et al. [14]. Briefly, after centrifugation of the bacterial suspensions and collecting the pellets, they were resuspended in saline and transferred to MHB, then they were incubated for only 1 h at 37 °C. The bacterial suspensions then were centrifuged and the pellets were resuspended in the phosphate urea magnesium sulfate buffer (PUM buffer, pH 6.9). Different volumes of n-hexane, ranging from 0.3 to 1.8 ml, were added to 4.8 ml of bacterial suspension in PUM buffer, thoroughly mixed for 2 min, and after complete phase partition, the aqueous phase was taken and its absorbance was measured at 540 nm. The hydrophobicity index (HI) was calculated using the following formula:
$$ HI=\left(A540 control-A540 test\right)/A540 control $$
Impact of adaptation on biofilm formation using crystal violet assay
Biofilm formation of the tested isolates before and after BAC adaptation was assessed as described by Yang et al. [15]. After overnight incubation of the tested bacteria, its OD600 was adjusted at 1.0, and they were diluted using sterile brain heart infusion (BHI) broth (Oxoid, UK) to a ratio of 1:100. Then, 100 μL of each bacterial suspension was seeded in 96-well microtitration plates, utilizing 3 wells for each isolate, and the wells that contained BHI broth alone were the negative control. After 24 and 48 h of incubation at 37 °C, the wells were washed 3 times with water. The formed biofilms were then fixed with 100 μL of methanol and left for 20 min and air-dried for 1 h. The fixed bacteria were stained with crystal violet solution and left for 10 min, then washed 5 times with water and air-dried. Finally, the dye was solubilized using 100 μl acetic acid solution and OD490 was measured by ELISA AutoReader (Sunrise Tecan, Austria).
The tested isolates were classified into four groups based on their ability to form a biofilm (their ODs) as follows:
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a-
NBP: not biofilm producer (ODc < OD < 2 ODc),
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b-
WBP: weak biofilm producer (2 ODc < OD < 4 ODc),
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c-
MBP: moderate biofilm producer (4 ODc < OD < 6 ODc) and
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d-
SBP: strong biofilm producer (6 ODc < OD).
The cut-off OD (ODc) of the microtitration plate is three standard deviations above the mean OD of the negative control.
Determination of the viability of microbial cells inhabiting the biofilm population
The MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) method that is based on the reduction of a tetrazolium salt was accomplished using the method previously described [16]. In brief, the bacterial suspension was incubated in 96-well microtitration plates and the free-floating cells were removed. About 150 μl of phosphate-buffered saline (PBS) containing 50 μl of MTT solution, at a concentration of 0.3%, were added to each well and incubated for 2 h at 37°C. The MTT solution was then withdrawn from the wells and 150 μl of dimethyl sulphoxide (DMSO) was added and incubated at room temperature for 15 min. The absorbance of the resulting solution was measured using ELISA AutoReader at a wavelength of 550 nm.
RT-PCR
Detection of the relative gene expression of the biofilm specific gene using Rotor-Gene Q 5 plex apparatus (Qiagen, Germany) was accomplished before and after adaptation of 10 K. pneumoniae isolates to BAC utilizing the Rotor-Gene Q software. The gene gapA was utilized as the housekeeping gene [17]. Total RNA was extracted according to the protocol of the manufacturer of Purelink® RNA Mini Kit (Thermo Scientific, USA). RNA was then converted into cDNA using the power cDNA synthesis kit (first-strand cDNA synthesis) (iNtRON Biotechnology, Korea). The amplification of the biofilm gene (bssS) was carried out using Power SYBR® Green master mix (Thermo SCIENTIFIC, USA). Primers designed as previously described by Hassan et al. [18] with the following sequences: the forward primer 5′-GATTCAATTTTGGCGATTCCTGC-3′ and the reverse primer 5′-TAATGAAGTCATTCAGACTCATCC-3′. Relative gene expression was calculated by the 2−ΔΔCt method [19], and the isolates before BAC adaptation were utilized as control samples. When there was an increase of 2-fold or more in comparison with that of the control samples, overexpression was designated (Fernández-Cuenca et al. 2015). All the experiments were performed in triplicate and the result values were expressed as mean ± SD.
PCR and nucleotide sequencing
The genomic DNA was extracted using the GeneJET Genomic DNA Extraction Kit following the manufacturer’s protocol (Thermo Scientific, USA). Amplification using the forward and reverse primers of the biofilm gene bssS (mentioned before) was performed in a total volume of 25 μl containing 1 μl DNA extract, 1 μl of forward primer (10 μM), 1 μl of reverse primer (10 μM), 12.5 μl of Dream green PCR master mix (Thermo Scientific, USA) and 9.5 μl of nuclease-free water. The thermocycler (Thermo Scientific, USA) was programmed with the following conditions including initial denaturation at 94 °C for 2 min, followed by 35 cycles of denaturation at 94 °C for 40 s, annealing at 48 °C for 1 min, extension at 72 °C for 1 min, and final cycle of amplification at 72 °C for 5 min. For the conformation of production of PCR product, the generated amplicon (225 bp) was visualized on 1.5% agarose gel electrophoresis stained with ethidium bromide and illuminated under UV transilluminator (Kowell, Spain). The PCR amplicons were purified using the PCR Purification Kit (PP-201XS; Jena Bioscience, Germany) according to the manufacturer’s instructions. The purified PCR products were then sent to LGC Company (Germany) to be sequenced in the forward direction using ABI 3730XL DNA sequencer (Thermo SCIENTIFIC, USA).
Bioinformatic analysis
The sequences were analyzed using the Chromas 2.6.5 program (https://technelysium.com.au/wp/chromas/) and the identity of the sequenced PCR products was examined using Blast search against GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The alignments and assembly of the sequences were performed using Jalview 2.10.2 software.
Statistical analysis
Independent repeating of experiments (at least three times) was carried out in order to achieve reproducibility. The expression of the data was in the form of mean and standard deviation. One-way ANOVA was utilized to detect the significant differences among the tested groups (p < 0.05) using IBM SPSS (17.0, IBM, USA).
