We engineered a CRISPR interference (CRISPRi) system targeting the AcrAB-TolC efflux pump to prevent MDR development in Escherichia coli. Nine specific single-guide RNAs (sgRNAs) were designed to target the components of the AcrAB-TolC efflux pump, namely AcrA, AcrB and TolC. A total of thirteen CRISPRi recombinant plasmids were constructed with single or clustered sgRNAs. The transcriptional levels of the target genes, MICs of multiple antibiotics and biofilm formation in each CRISPRi strain were tested. The CRISPRi system expressing sgRNA clusters targeting acrB and tolC simultaneously exhibited the highest inhibitory effect on AcrAB-TolC efflux pump activity in E. coli HB101, with 78.3%, 90.0% and 65.4% inhibition rates on the transcriptional levels of acrA, acrB and tolC, respectively. The CRISPRi system resulted in ∼2-, ∼8- and 16-fold increased susceptibility to rifampicin, erythromycin and tetracycline, respectively. In addition, the constructed CRISPRi system reduced biofilm formation with inhibition rates in the range of 11.2% to 58.2%. To the best of our knowledge, this is the first report on the construction of an inducible CRISPRi system targeting the AcrAB-TolC efflux pump to prevent MDR development in E. coli. This study provides insights for future regulation and manipulation of AcrAB-TolC activity and bacterial MDR by a CRISPRi system.

Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic-resistant genes (ARGs) among human pathogens. The spread of ARGs can be halted or diminished by interfering with the conjugation process. In this study, we explored the possibility of using an rbsB gene as a single target to inhibit plasmid-mediated horizontal gene transfer in Escherichia coli by CRISPR interference (CRISPRi) system. Three single-guide RNAs (sgRNAs) were designed to target the rbsB gene. The transcriptional levels of the rbsB gene, the conjugation-related genes, and the conjugation efficiency in the CRISPRi strain were tested. We further explored the effect of the repressed expression of the rbsB gene on the quorum sensing (QS) system and biofilm formation. The results showed that the constructed CRISPRi system was effective in repressing the transcriptional level of the rbsB gene at a rate of 66.4%. The repressed expression of the rbsB gene resulted in the reduced conjugation rate of RP4 plasmid by 88.7%, which significantly inhibited the expression of the conjugation-related genes (trbBp, trfAp, traF and traJ) and increased the global regulator genes (korA, korB and trbA). The repressed rbsB gene expression reduced the depletion of autoinducer 2 signals (AI-2) by 12.8% and biofilm formation by a rate of 68.2%. The results of this study indicated the rbsB gene could be used as a universal target for the inhibition of conjugation. The constructed conjugative CRISPRi system has the potential to be used in ARG high-risk areas.

We explored the antimicrobial activity of sertraline on Listeria monocytogenes and further investigated the effects of sertraline on biofilm formation and the virulence gene expression of L. monocytogenes. The minimum inhibitory concentration and minimum bactericidal concentration for sertraline against L. monocytogenes were in the range of 16–32 μg/mL and 64 μg/mL, respectively. Sertraline-dependent damage of the cell membrane and a decrease in intracellular ATP and pH_in in L. monocytogenes were observed. In addition, sertraline reduced the biofilm formation efficiency of the L. monocytogenes strains. Importantly, low concentrations (0.1 μg/mL and 1 μg/mL) of sertraline significantly down-regulated the expression levels of various L. monocytogens virulence genes (prfA, actA, degU, flaA, sigB, ltrC and sufS). These results collectively suggest a role of sertraline for the control of L. monocytogenes in the food industry.

Tigecycline is a last-resort antibiotic for the treatment of infections caused by multidrug-resistant bacteria. The emergence of plasmid-mediated tigecycline resistance genes is posing a serious threat to food safety and human health and has attracted worldwide attention. In this study, we characterized six tigecycline-resistant Escherichia fergusonii strains from porcine nasal swab samples collected from 50 swine farms in China. All the E. fergusonii isolates were highly resistant to tigecycline with minimal inhibitory concentration (MIC) values of 16–32 mg/L, and all contained the tet(X4) gene. In addition, 13–19 multiple resistance genes were identified in these isolates, revealed by whole-genome sequencing analysis. The tet(X4) gene was identified as being located in two different genetic structures, hp-abh-tet(X4)-ISCR2 in five isolates and hp-abh-tet(X4)-ΔISCR2-ISEc57-IS26 in one isolate. The role of efflux pumps in tigecycline resistance was evaluated by using inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The MIC values of tigecycline showed a 2- to 4-fold reduction in the presence of CCCP, indicating the involvement of active efflux pumps in tigecycline resistance in E. fergusonii. The tet(X4) gene was found to be transferable to Escherichia coli J53 by conjugation and resulted in the acquisition of tigcycline resistances in the transconjugants. Whole-genome multilocus sequence typing (wgMLST) and phylogenetic analysis showed a close relationship of five isolates originating from different pig farms, suggesting the transmission of tet(X4)-positive E. fergusonii between farms. In conclusion, our findings suggest that E. fergusonii strains in pigs are reservoirs of a transferable tet(X4) gene and provide insights into the tigecycline resistance mechanism as well as the diversity and complexity of the genetic context of tet(X4) in E. fergusonii.