Cells and bacteria
The human respiratory epithelial cells (HPAEpiC,Cat. No. 3200) were purchased from ScienCell Research Laboratories, Inc. (San Diego, California) and maintained in RPMI 1640 medium containing 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (GIBCO) at 37 °C and 5% CO2. The use of HPAEpiC cells can better reflect the colonization or infection of Acinetobacter baumannii in human respiratory tract and is close to the human environment. The clinical isolate of multidrug-resistant Ab (Additional file 5: Table S2) was originally collected from the sputum of patient hospitalized in Hangzhou First People’s Hospital, China. After identification of genome sequencing (Sangon Biotech Co., Ltd., Shanghai; the SRA accession: PRJNA523637), the bacteria were inoculated to blood agar plates for 18-20 h at 37 °C, and then single colonies were picked to suspend in 0.45% sodium chloride solution for preparing bacterial suspension. The bacteria per ml was calculated based on 1 McF = 2 × 108 CFU/ml.
Host-bacterial co-culture
At different MOIs
The range of bacterial MOIs was selected according to previous studies[15]. A 1 ml aliquot of HPAEpiC (2 × 105 cells/ml) was seeded into six-well plates containing RPMI 1640 medium. Immediately, 100 μl of different concentrations of bacteria (2 × 106, 2 × 107, 1 × 108 and 2 × 108CFU/ml, corresponding to the MOIs 1, 10, 50 and 100, respectively) were inoculated into each well, and 0.45% NaCl was used as the normal control. The effects of Ab on the morphology and proliferation of epithelial cells at different MOIs and co-culture time points (2 h, 4 h, 6 h and 8 h) were observed by an IX70 bright field inverted microscopy (Olympus Optical Co., Ltd., Japan) at a magnification of 30×. Meanwhile, we identified the live and dead epithelial cells according to the LIVE-DEAD viability/cytotoxicity assay kit instructions (Life Technologies, Grand Island, NY, USA) and detected using an IX71 inverted microscopy with Nomarski optics (Olympus Optical Co., Ltd., Japan). Based on these observations, a suitable time period for the host-bacterial co-culture was selected for subsequent experiments.
The liquid in each well of the six-well plates was carefully discarded at the end of the indicated host-bacterial co-culture time period. Before digestion with trypsin solution for 1-2 min at 37 °C, the cells were washed once with phosphate buffer saline (PBS). The cells were then harvested by centrifugation (100 × g, 5 min) and washed twice with PBS. Finally, bacterialDNA was extracted from 1 × 105 cells based on procedures described by Chen et al[26]. qPCR was performed with SYBR Premix Ex Taq II (TaKaRa Bio Inc., Shiga, Japan) on an ABI 7500 real-time PCR instrument (Applied Biosystems, United States) according to the manufacturer’s instructions. Each test (50-μl volume) was performed in triplicate. Sterile distilled water served as a negative control, and bacterialDNA served as a positive control. Primer sequences for the target gene (ompA, outer membrane protein A) were as follows: 5'-CACAGATAACACTGGTCCACG-3' and 5'-GAATACACGACGGTTCATAGC-3'. The changes in Ab invasion of epithelial cells (including strong adhesion) at different MOIs were anaylsed by comparing the Ct values obtained.
Detailed RTCA (real time cellular analysis) experimental procedures have been previously described[15, 19, 20]. Briefly, 50 μl of medium was added to 16-well E-plates (specific cell culture plates for RTCA) to obtain background readings, which were followed by the addition of 100μl of a cell suspension (2×105 cells/ml). After the E-Plate was incubated at room temperature for 30 min, and 10 μl of different concentrations of bacterial suspension or 0.45% NaCl was added to the wells containing cells. The E-Plates were placed onto the reader in the incubator for continuous recording of the CI. The cells were monitored every 5 min for 72 h to obtain TCRPs. The data were collected from three multiple-well duplicates and presented as the CI normalized (CI = 1.00) to the last time point before intervention. Additionally, a suitable bacterial concentration was selected for subsequent experiments based on the above results.
In different states of cell incubation
The purpose of this experiment was to observe the effect of Acinetobacter baumannii on cell proliferation under different cell culture conditions and cell adherent growth conditions. In other words, what kind of cell incubation (cell suspension, cell partial adherent fusion, cell complete adherent fusion) is more suitable to add bacterial suspension to cell petri dish (including cell and cell culture medium) to study host-bacterial interaction. This experiment was divided into three groups of different cell incubation states.
Cell suspension: the purpose of this experimental group was to observe the effect on the adhesion of Ab to host cell under initial suspension state of cell suspension added to six-well plates (0 h); 30-40% cell confluence: the purpose of this experimental group was to observe the effect of Acinetobacter baumannii on epithelial cells without growth space restriction (The cells have been cultured for 24 hours); 80-90% cell confluence: the purpose of this experimental group was to observe the effect of Acinetobacter baumannii on epithelial cells under the cell incubation state of 80%-90% cell confluency after 48 hours of cell culture, this state was close to the human environment.
A total of 2 ml of cells (2 × 105 cells/ml) were seeded into six-well plates containing RPMI 1640 medium. 200 μl of the indicated concentration of bacteria was added to wells containing cells in different incubation states (cell suspension, 30-40% cell confluence and 80-90% cell confluence) and 0.45% NaCl was used as the normal control. An inverted microscopy was used to observe the dynamic process of the interaction between Ab and epithelial cells at different states of cell incubation (2 h, 4 h, 6 h, 8 h and 24 h).
RTCA experimental procedures were as described above. A total of 10 μl of the above mentioned bacterial suspensions, 0.45% NaCl or sterile distilled water was added to wells containing cells at different incubation states (cell suspension, 30-40% cell confluence and 80-90% cell confluence). Wells without intervention were used as the blank control. The interaction between Ab and human respiratory epithelial cells were evaluated based on the TCRP results and microscopic imaging.
Effect of calcium on host-bacterial interaction
Ab growth assays
A total of 400 μl of each of the indicated concentrations of bacteria and different concentrations (12 mmol/L, 24 mmol/L, 36 mmol/L and 48 mmol/L) of a calcium chloride solution (CaCl2) were added to wells containing 2 ml of RPMI 1640 medium. A 400 μl aliquot of each of the bacterial suspensions and EDTA solution (5 mmol/L) was used as the normal control I (taking into account the presence of calcium in RPMI, the final calcium concentrations in experimental groups were 1.4 mmol/L, 2.4 mmol/L, 3.4 mmol/L and 4.4 mmol/L, respectively, and the normal control I was 0 mmol/L). The effect of different concentrations of calcium and culture times (2 h, 4 h, 6 h, 8 h, 16 h and 24 h) on the proliferation of Ab were measured at 600 nm using a microplate reader (Sunrise, Tecan, Switzerland). Each experiment was performed in triplicate.
Experiments on the differential expression of Ab-related genes
The sequences of Ab-related genes were selected according to previous studies[12, 16, 27, 28]. Specifically, the recA (internal reference), ompA(outer membrane protein A),bfmRS(two-component regulatory system) andabaI (autoinducer synthase) sequences were downloaded from GenBank. The primers (Table 2) for reverse transcription quantitative PCR (RT-qPCR) were designed using the Primer Premiers 5.0 software, selected based on a BLAST sequence comparison, and synthesized by Life Technology (Shanghai, China). After incubation for the indicated time period described above, all bacteria were collected and total RNA was isolated from each group using an AxyPrep Miniprep Kit (Corning Inc., NY, USA) according to the manufacturer’s instructions. The concentration and purity (OD260/OD280 of approximately 2.0) of the RNA samples were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). RT-qPCR was performed with the One Step SYBR PrimeScript PLUS RT-PCR Kit (TaKaRa) on an ABI 7500 real-time PCR instrument according to the manufacturer’s instructions. Each test (50 μl volume, 20 ng of RNA template) was performed in triplicate. RNase free distilled water served as negative control I and templates treated with RNase served as negative control II. The differential expression of Ab-related genes (ompA, bfmRSand abaI) under different calcium concentrations was anaylsed by comparing the resulting Ct values.
Ab biofilm assays
Mutant strains of Ab deficient in ompA (Ab-ompA-) were constructed and modified according to the conjugative transfer and parental conjugation methods[29, 30]. Briefly, a modified pMO130-TelR with the fragments corresponding to the regions up and downstream of the ompA gene generated from genome of Abwas constructed. The resultant plasmid was then transformed into theE.coli S17-1 λ pir competent cells. Then, trans-conjugation was performed between E.coli S17-1 λ pir donor strain and Ab recipient strain to transfer and integrate pMO130-TelR-ompA (Up/Down) into the chromosome of Ab.Donor and recipient strains were plated onto LB agar containing tellurite (30 mg/L) and gentamicin (25 mg/L) and incubated at 37 °C overnight. 0.45 mol/L catechol solution was sprayed on the surface of the plate, and yellow clones were picked for PCR identification. The second selection was then performed by incubating Ab on 10 % sucrose with salt-free LB agar plates, stayed overnight at 37 °C to identify sucrose-resistant sensitive clones, then analyzed by PCR and sequencing to confirm that the target gene was excised, resulting in an unmarked in-frame deletion. A single clone with no ompA sequence was saved as Ab-ompA-. Ab and Ab-ompA- isolates were added separately to 96-well plates containing RPMI 1640 medium of different calcium concentrations (range from 0 to 4.4 mmol/L). The biofilm formation was measured based on procedures outlined by Selasiet al.[31] with some modifications. After 24h, the liquid in each well was carefully discarded and washed three times with PBS. Then, the plates were air-dried and stained with crystal violet (0.1% v⁄v) for 20 min. Finally, the plates were washed three times with PBS, air-dried and decolorized with ethanol (95% v⁄v) for 20 min. The turbidity was measured at 580 nm using the Tecan Sunrise microplate reader. Additionally, the turbidity was also measured at 600 nm before biofilm mass staining to compensate for bacterial growth differences. Biofilm formation was quantified by calculating the ratio of OD580/OD600. Each experiment was performed in triplicate.
Experiments on the morphology and proliferation of epithelial cells
A 2 ml cell suspension (2 × 105 cells/ml) was seeded into six-well plates and allowed to attach and grow for 48 h to reach the platform stage (80-90% cell confluence) before the addition of a 400 μl aliquot of each of sterile distilled water and different concentrations of CaCl2. Additionally, a 400 μl aliquot of each of sterile distilled water and EDTA was used as the normal control. The effects of different concentrations of calcium and culture times (2 h, 4 h, 6 h, 8 h and 24 h) on the morphology and proliferation of epithelial cells were observed by inverted microscopy.
RTCA experimental procedures were as described above. After incubation for 48 h, cells were treated with a 20 μl aliquot of each of sterile distilled water and different concentrations of CaCl2 or a 20 μl aliquot of each of sterile distilled water and EDTA. A well without intervention was used as a blank control. The effects of calcium on the morphology and proliferation of epithelial cells were evaluated based on the TCRP results and microscopic imaging.
Experiments on host-bacteria interactions
A 2 ml cell suspension (2 × 105 cells/ml) was seeded into wells and allowed to attach and grow for 48 h before the addition of a 400 μl aliquot of each of the indicated bacterial concentrations and different concentrations of CaCl2. Additionally, a 400 μl aliquot of each of the bacterial suspensions and EDTA was used as the normal control I. The effect of Ab on the morphology and proliferation of epithelial cells with different calcium concentrations and co-culture times (2 h, 4 h, 6 h, 8 h and 24 h) were observed by inverted microscopy.
According to the procedures described above, total bacterial RNA was isolated from each group and RT-qPCR was performed after host cells and bacteria were co-cultured for the indicated time period. The expression of Ab-related genes in the biotic environment under different calcium concentrations was anaylsed by comparing the Ct values obtained.
In addition, a 400 μl aliquot of each of sterile distilled water and 0.45% NaCl was used as the normal control II. All the liquid in the wells was carefully discarded after a 2 h, 4 h or 6 h incubation, as described above. Before digestion, the cells were washed three times with PBS. Other procedures, including bacterial DNA extraction and qPCR were carried out as described above. The changes of Ab invasion to epithelial cells (including strong adhesion) under different calcium concentrations and co-culture times was anaylsed by comparing the Ct values obtained.
RTCA experimental procedures were as described above. After the cells (20,000 cells per well) were incubated for 48 h, the two part experiment was carried out as follows:
- A 20 μl aliquot of each of the abovementioned bacterial suspensions and different concentrations of CaCl2 was added to the wells; the bacterial suspensions and EDTA was used as the normal control I; sterile distilled water and 0.45% NaCl was used as the normal control II.
- A 20 μl aliquot of each of different concentrations of CaCl2 and the bacterial suspension/0.45% NaCl/sterile distilled water was added to the wells; a total of 20 μl of sterile distilled water was added as the normal control. Wells without intervention were used as the blank control.
The effect of calcium on the host-bacterial interactions was evaluated based on the TCRP results, qPCR and microscopic imaging.
Statistical analysis
The data are presented as the means ± SD. One-way analysis of variance (ANOVA) and the Student-Newman-Keuls (SNK) test were applied to compare and analyse the changes in Ct values at different MOIs and the effects of calcium on the expression of Ab-related genes. Two-way ANOVA and the SNK test were applied to analyse the effects of calcium on biofilm formation of different Ab strains. The effects of different calcium concentrations and culture times on the CI of epithelial cells were analysed using multivariate ANOVA with repeated measures and the SNK test. Univariate ANOVA with repeated measures and the SNK test were used to compare and analyse the effects of different calcium concentrations and co-culture times on Ab invasion (including strong adhesion) of epithelial cells (Ct values). P< 0.05 was considered to be statistically significant.