Objectives We engineered a CRISPR interference (CRISPRi) system targeting the AcrAB-TolC efflux pump to prevent MDR development in Escherichia coli. Methods 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. Results 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%. Conclusions 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.

Salmonella enterica resistant to colistin, third-generation cephalosporins (3GCs), and fluoroquinolones (FQs) has been deemed a high-priority pathogen by the World Health Organization (WHO). The objective of this study was to characterize 11 mcr-1-harboring Salmonella enterica serovar Typhimurium isolates from raw pork and ready-to-eat (RTE) pork products in Guangzhou, China. All isolates were multi-drug resistant and contained 6–24 antibiotic-resistant genes. The mcr-1 gene was localized in the most conserved structure (mcr-1-orf ) in eight isolates and in mobile structure (ISApl1-mcr-1-orf ) in three isolates. One raw pork isolate SH16SF0850, co-harbored mcr-1, blaCTX−M−14, and oqxAB genes. One isolate 17Sal008 carried mcr-1, blaCTX−M−14, qnrS2, and oqxAB genes located on a 298,622 bp IncHI2 plasmid pSal008, which was obtained from an RTE pork product for the first time. The pSal008 was closely related to a plasmid in an S. typhimurium isolate from a 1-year-old diarrheal outpatient in China and was found to be transferable to Escherichia coli J53 by conjugation. Genome sequence comparisons by core-genome Multi Locus Sequence Typing (cgMLST) based on all S. typhimurium isolates from China inferred highly probably epidemiological links between selected pork isolates and no possible epidemiologically links between RTE pork isolate 17Sal008 and other isolates. Our findings indicate that raw pork and pork products are potential reservoirs of mcr-1-harboring S. typhimurium and highlight the necessity for continuous monitoring of colistin, 3GCs, and FQs resistant S. typhimurium from different origins.

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.

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