Specimen source

This study used 35 strains of A. baumannii, comprising 10 strains categorized as CSAB and 25 strains classified as CRAB, including those with levofloxacin resistance.

Instruments and reagents

The main instruments used in the study included a mold incubator (MJX-160B-Z; Shanghai Boxun Industrial Co., Ltd.), a biological safety cabinet (BSC-1600II.B2; Shanghai Sujing Industrial Co., Ltd.), a bacterial turbidity meter (WGZ-2XJ; Shanghai Xinrui Instrument Co., Ltd.), and a Piko Real real-time PCR instrument (TCR0024; Thermo Fisher Scientific, USA).

The main reagents used were 2×RealStar Power SYBR qPCR Mix (GeneStar) and imipenem and levofloxacin (Shanghai Yuanye Biotechnology Co., Ltd.).

Server and tools

This study conducted all bioinformatics analyses using SWISS-MODEL (https://swissmodel.expasy.org/) and the PubChem database (http://pubchem.ncbi.nlm.nih.gov/). Molecular docking was performed using Autodock, and visualization of docking complexes was performed using Pymol.

Preparation of receptor proteins

Receptor proteins were prepared by querying the protein sequences of Bae S and Bae R from the National Center for Biotechnology Information (NCBI) database. The entry sequences were WP_012300504.1 (https://www.ncbi.nlm.nih.gov/protein/WP_012300504.1) and WP_000680574.1 (https://www.ncbi.nlm.nih.gov/protein/WP_000680574.1), respectively. The secondary structure was predicted using the PSIPRED database (http://bioinf.cs.ucl.ac.uk/psipred/), whereas the NetPhos-3.1 server (https://services.healthtech.dtu.dk/) was used to predict serine, threonine, and tyrosine sites. The SWISS-MODEL was used to build the three-dimensional (3D) structure models of Bae S and Bae R. Template sequences numbered 7CCH (https://www.rcsb.org/structure/7CCH) 4B09 (https://www.rcsb.org/structure/4B09) were forecasted, and PDB files were downloaded. Autodock software was used for hydrogenation, charge calculation, and atomic type setting of receptor proteins. In addition, the VADAR 1.8 online server (http://vadar.wishartlab.com/) was used to calculate the statistical percentage of protein secondary structure, generate the Ramachandran graph [12], evaluate the percentage values of the optional and preferred regions in the graph.

Preparation of small-molecule ligands

The 3D structures of imipenem, meropenem, and levofloxacin were retrieved from the PubChem database using their respective PubChem IDs: 104,838 (https://pubchem.ncbi.nlm.nih.gov/compound/104838), 441,130 (https://pubchem.ncbi.nlm.nih.gov/compound/441130), and 149,096 (https://pubchem.ncbi.nlm.nih.gov/compound/149096). The structures were downloaded in SDF file format. Autodock software was used to process small-molecule ligand charge adjustments.

Generation of receptor proteins grid and molecular docking

Specific grid cube box sizes were entered with fixed values for the x, y, and z axes to predict the phosphorylation sites as docking pockets. For Bae S, the grid box was fixed at X = 19.374, Y = 67.897, and Z = 4.26, whereas for Bae R, the grid box values were fixed at X = − 21.361, Y = − 67.564, and Z = − 80.515. Autodock software was used to dock the receptor protein and small-molecule ligand. Different molecular docking complexes were analyzed based on their binding energy values (kcal/moL) and hydrogen bonds. Visual results were further analyzed using Pymol.

Determination of MIC values and generation of time–sterilization curve

A single colony was inoculated into LB liquid medium and incubated at 200 rpm and 37℃ to the logarithmic growth phase. For each well of a sterile 96-well plate,100 µL of LB liquid medium and 10 µL of overnight cultured bacterial liquid were added. Then, 100 µL of imipenem solution was added for gradient dilution to achieve final concentrations of 128, 64, 32, 16, 8, 4, 2, 1 µg/mL. The plate was then cultured at 37℃ for 18–24 h, and the bacterial growth was observed by the naked eye to determine the MIC as the lowest concentration inhibiting visible growth. In parallel, antibiotics at a final concentration of ½ MIC were added to the bacterial solution. The mixture was then incubated at 200 rpm and 37 °C on a shaker. Samples were collected at 0, 2, 4, 6, 12, and 24 h post-administration. Then, 100 µL of bacterial solution extracted each time was diluted 10 times with 0.9% normal saline 4 times, resulting in a final dilution of 1:10,000. Then, 200 µL of the diluted bacterial solution was spread onto an LB solid medium and mixed thoroughly. The plates were inverted and incubated at 37℃ for 18–24 h, and the colonies were counted. A time–sterilization curve was plotted using the administration time as the horizontal coordinate (x-axis) and the logarithm of the colony-forming units as the vertical coordinate (y-axis).

Primer design and reverse transcription PCR detection of relative expression levels of two-component system genes bae S and bae R

RNA was extracted using the traditional Trizol method, followed by reverse transcription to obtain cDNA. The 16S rRNA gene was used as the internal reference gene. The Ct values of the two-component system genes bae S and bae R were determined using fluorescence quantitative PCR. The reaction conditions included an initial denaturation at 95℃ for 10 min, followed by denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min, and 40 cycles of amplification(refer to the instructions for GeneStar(https://www.gene-star.com/) A311 product). The formula used for calculating relative expression was as follows: ΔCt = Cttarget gene – Ctinternal reference gene; ΔΔCt = ΔCtexperimental group – ΔCtcontrol group; and gene expression = 2–ΔΔCt. The experiment was conducted in triplicate.

Nucleotide sequence registration number

The nucleotide sequences of the required genes in this study were stored in the NCBI database under the GenBank accession numbers: 16S rRNA (LN611374.1), bae S (MK344183.1), and bae R (MK344168.1). The sequence of primers used in this study is shown in Table 1.

Table 1 Primer sequences used in this studyStatistical analysis

Each sample was replicated three times, and statistical analysis was performed using SPSS 22.0 software. A bar chart was created using GraphPad Prism 9 software. Two independent-samples t tests were used to compare the mRNA expression levels between the CRAB and the CSAB groups. A P value < 0.05 indicated a statistically significant difference, a P value < 0.01 indicated a significant difference, and a P value < 0.001 indicated an extremely significant difference.