The strain information was listed in Table 1. The derivative A. veronii strains included wild-type, Δhfq, Δhfq:hfq, and Δhfq:acrAB. The strains were cultured in M9 minimal medium (M9) at 30^°C, 150 r/min, supplemented with 50 μg/mL ampicillin. Δhfq represents the hfq knockout strain (Zhang et al., 2019). Δhfq:acrAB overexpresses the acrAB gene in the hfq knockout strain. The strain E. coli WM 3064 was applied to assist in the introduction of the plasmids into A. veronii by tri-parent conjugation (Simon et al., 1983; Ferrieres et al., 2010). For the culture of strain WM3064, 0.3 mM diaminopimelic acid was supplemented in LB at 37^°C.

The vector and primers were listed in Table 2. For the construction of the expression vector of the efflux pump-associated gene acrAB, the acrA and acrB genes were inserted into the plasmid pBBR1MCS-2, wherein the enzyme cleavage sites were SalI and EcoRI. The upstream and downstream primers required for the construction were acrA F 5′-ACGCGTCGACTTGGTATCGGCTGGGGATTG-3′ and acrB 5′-CCGGAATTCATGAGCGTCGGGAGAG-3′.

Antibiotics were added to sterile 96-well plates at final concentrations of 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg/mL (Andrews, 2001). Then, 10⁶ CFU broth was added to each well to a final volume of 200 μL. The 96-well plate was sealed with parafilm and cultured at 30^°C with shaking at 150 r/min for 24 h. The experiment was repeated for 3 times.

The wild type and Δhfq strain were cultured in M9 medium containing 50 μg/mL ampicillin, cultured at 30^°C and 150 r/min for 20 h, centrifuged to remove the culture medium, and washed with sterile PBS for transcriptomic analysis. The sequencing was carried out by BGI (Beijing Genomics Institution). The cells were collected and lysed, and the sample RNA was extracted with phenol-chloroform. The concentration and quality of the RNA samples were tested with the Agilent 2100. DNase I was used to remove double-stranded DNA, and a Ribo-Zero Magnetic Kit was used to remove ribosome RNA. Reverse transcription was performed with random primers and first strand cDNA as a template to synthesize the second strand. The linker sequence was attached to the 3′ end of the cDNA fragment. The cDNA sequence was amplified with a primer cocktail, and the purified product was sequenced on a HiSeq Xten (Illumina, San Diego, CA, United States) platform. The sequencing depth was chain-specific sequencing for 2 Gb of clean data. HISAT was attempted for genome assembly, potential coding sequence analysis and new transcript identification that may be present. The transcriptional differences between wild-type and hfq knockout were analyzed by Bowtie 2, and FPKM was used to normalize gene expression levels. Each gene expression was calculated using the Benjamini-Hochberg false discovery rate (FDR). The differential transcripts were tested for log-fold change, and the p value was corrected with FDR < 0.001. The differential genes were analyzed using GO classification, and disparity expression in the pathway was compared with the entire genomic background using hypergeometric analysis p ≤ 0.05 was a differential metabolic pathway. GEO accession number was GSE120603, and the URL of accession website was displayed as https://submit.ncbi.nlm.nih.gov/subs/sra/SUB6133286. The DESeq. 2 packages in R were applied to estimate the fold changes and perform other analysis. A. veronii TH0426 genome (Genomic Sequence: NZ_CP012504.1) was referenced for transcriptome analysis (Kang et al., 2016).

The non-target metabolomic and lipidomic detection platform (UHPLC-QTOF-MS) was applied to metabolomics for the detection of A. veronii samples. UHPLC-QTOF-MS included Ultra-Performance Liquid Chromatography 1290UHPLC (Agilent), ACQUITY UPLC BEH Amide column 1.7 μm, 2.1 × 100 mm (Waters) and High-Resolution Mass Spectrometry Triple TOF 6600 (AB Sciex). The original mass spectrum was converted to the mzXML format using Proteo Wizard software, and the peaks were identified using the R Programming Language package (Version 3.2) and self-built secondary mass spectrometry data. URL of accession website was displayed as www.ebi.ac.uk/metabolights/MTBLS1411.

Statistical data were analyzed using the statistical Package for the Social Science (SPSS) version 20.0 (SPSS, Chicago, IL, United States) and GraphPad Prism version 8.0 (GraphPad, San Diego, CA, United States). The results are presented as the mean values of three independent experiments with standard deviation using one-way analysis of variance. p < 0.05 or 0.01 were represented as significant or extremely significant, respectively.

According to previous studies, A. veronii were resistant to gentamycin, kanamycin, streptomycin, and were sensitive to chloramphenicol, ciprofloxacin (Liu et al., 2018). Trimethoprim, as an antibiotic that inhibits folic acid metabolism, has a strong inhibitory effect on a variety of bacteria. Resistance to trimethoprim was found to be altered in the absence of hfq (Figure 1A). The mutant Δhfq was more sensitive to trimethoprim than wild type, which exhibited with a minimum inhibitory concentration (MIC) of 8 μg/mL in contrast to 16 μg/mL of wild type. The complemented strain attenuated the sensitivity of Δhfq to trimethoprim, and the MIC was the same as that of wild type (Figure 1B).

To understand the changes of drug resistance in Δhfq strains, transcriptome sequencing was used to compare metabolic pathways with significant variations in expression levels and to analyze their relationship with trimethoprim resistance. The clustering analysis revealed that many genes related to purine metabolism and efflux pump synthesis were expressed differently (Figure 2). Although the direct target of trimethoprim was dihydrofolate reductase, the transcriptions of dihydrofolate reductase were not significantly different between Δhfq and wild type (Figure 2 marked with star). However, the expression of 53 genes was affected in purine metabolism, which functioned as the downstream of folate metabolism (Figure 3). There were 21 genes marked with red were up-regulated which led to purine accumulation. Two genes marked with green, as purine consuming enzymes including xanthine nucleic acid transferase and hypoxanthine nucleic acid transferase, were significantly down-regulated.

Due to the significant changes of transcription in metabolic pathways, variant metabolites of these pathways have been hypothesized to be responsible for trimethoprim resistance. The differential metabolites of wild-type and Δhfq were screened, analyzed, and classified into metabolic pathways (Figure 4). Purine metabolism, pyrimidine metabolism, and alanine, aspartate, and glutamic acid metabolism were greatly affected by the deletion of hfq, of which the effects on purine metabolism and pyrimidine metabolism were prominent. The deletion of Hfq incurred a significant increase of purine metabolites including adenosine, guanosine, and xanthine (Figure 5).

The purine metabolites, such as guanosine and adenosine, were significantly increased in Δhfq compared with wild type (Figure 3). To understand whether the accumulation of metabolites changed the trimethoprim sensitivity of hfq knockout, the downstream products such as 1 mM adenine, 1 mM guanine, and 1 mM ATP were added to the M9 medium to evaluate the MIC separately (Yang et al., 2019). The MIC of wild type was not altered when supplemented with 1 mM guanosine (Figure 6A), while that of Δhfq was decreased. There had little evident changes both in wild type and Δhfq after appending with 1 mM adenine or ATP. The above results suggested that the additional guanine enhanced the sensitivity of Δhfq to trimethoprim.

AcrAB-TolC is capable of actively transporting antibiotics (Li et al., 2015), and trimethoprim can be transported outside the cell membrane by an efflux pump of P. aeruginosa. According to the transcriptomic data, the mRNA levels of acrA and acrB in Δhfq were significantly reduced (by 9.30-fold and 9.34-fold) compared with wild type, but those of three copies of transcriptional repressor acrR, and that of component tolC (by 1.35-fold) were transcribed consistently (Figure 6B). The acrAB overexpression vector was constructed and transferred into the Δhfq strain. The overexpression strain Δhfq:acrAB showed an increased MIC and enhanced tolerance compared with Δhfq. Overexpression of acrAB reversed the loss of hfq, resulting in inefficient discharge of trimethoprim (Figure 6C).