Infections caused by antibiotic-resistant bacteria are a major public health threat. The emergence and spread of antibiotic resistance genes (ARGs) in the environment or clinical setting pose a serious threat to human and animal health worldwide. Horizontal gene transfer (HGT) of ARGs is one of the main reasons for the dissemination of antibiotic resistance in vitro and in vivo environments. There is a consensus on the role of mobile genetic elements (MGEs) in the spread of bacterial resistance. Most drug resistance genes are located on plasmids, and the spread of drug resistance genes among microorganisms through plasmid-mediated conjugation transfer is the most common and effective way for the spread of multidrug resistance. Experimental studies of the processes driving the spread of antibiotic resistance have focused on simple in vitro model systems, but the current in vitro protocols might not correctly reflect the HGT of antibiotic resistance genes in realistic conditions. This calls for better models of how resistance genes transfer and disseminate in vivo. The in vivo model can better mimic the situation that occurs in patients, helping study the situation in more detail. This is crucial to develop innovative strategies to curtail the spread of antibiotic resistance genes in the future. This review aims to give an overview of the mechanisms of the spread of antibiotic resistance genes and then demonstrate the spread of antibiotic resistance genes in the in vivo model. Finally, we discuss the challenges in controlling the spread of antibiotic resistance genes and their potential solutions.

The emergence and global epidemic of antimicrobial resistance (AMR) poses a serious threat to global public health in recent years. AMR genes are shared between bacterial pathogens mainly via horizontal gene transfer (HGT) on mobile genetic elements (MGEs), thereby accelerating the spread of antimicrobial resistance (AMR) and increasing the burden of drug resistance. There is an urgent need to develop new strategies to control bacterial infections and the spread of antimicrobial resistance. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are an RNA-guided adaptive immune system in prokaryotes that recognizes and defends against invasive genetic elements such as phages and plasmids. Because of its specifically target and cleave DNA sequences encoding antibiotic resistance genes, CRISPR/Cas system has been developed into a new gene-editing tool for the prevention and control of bacterial drug resistance. CRISPR-Cas plays a potentially important role in controlling horizontal gene transfer and limiting the spread of antibiotic resistance. In this review, we will introduce the structure and working mechanism of CRISPR-Cas systems, followed by delivery strategies, and then focus on the relationship between antimicrobial resistance and CRISPR-Cas. Moreover, the challenges and prospects of this research field are discussed, thereby providing a reference for the prevention and control of the spread of antibiotic resistance.

The emergence and spread of New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae have been a serious challenge to public health, and NDM-5 shows increased resistance to carbapenems compared with other variants. NDM-5 has been identified mostly in E. coli but has rarely been described in K. pneumoniae and other Enterobacteriaceae isolates. Here, we present the dissemination of highly similar 46-kb IncX3 bla _NDM-5 -carrying plasmids among multiclonal K. pneumoniae strains in children, highlighting the horizontal gene transfer of bla _NDM-5 among K. pneumoniae strains via the IncX3 plasmid. Moreover, the IncX3 bla _NDM-5 -carrying plasmids displayed strong stability in clinical strains when cultured in antibiotic-free medium, and the plasmid maintenance was attributed partly to conjugal transfer. Plasmid conjugation is mediated by the type IV secretion system (T4SS), and T4SS is conserved among all epidemic IncX3 bla _NDM-5 -carrying plasmids. Therefore, combining conjugation inhibition and promotion of plasmid loss would be an effective strategy to limit the conjugation-assisted persistence of IncX3 bla _NDM-5 -carrying plasmids.

Pseudomonas aeruginosa is a common opportunistic pathogen that causes acute nosocomial necrotizing pneumonia and is the predominant source of chronic lung infections in patients with the genetic disorder cystic fibrosis. Early diagnosis in infected patients and monitoring P. aeruginosa contamination is therefore of great importance in controlling disease spread and development with timely drugs intervention treatment and cut off infection source. Traditional culture-biochemical methods are time consuming and highly dependent on technicians and expensive instruments. To address these challenges, the present study aimed to develop a rapid, sensitive, and specific, on-site detection method for P. aeruginosa based on recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) technology. The experimental process included screening and modification of primer and probe sets targeting the unique virulence gene elastase B (lasB); specificity detection in 29 strains of P. aeruginosa and 23 closely-related pathogenic bacteria; sensitivity measurements with gradient-diluted P. aeruginosa genomic DNA and probit regression analysis; and clinical application evaluation using 574 patients samples and calculating coincidence rate and kappa index value in comparison with the culture-biochemical method. The P. aeruginosa RPA-LFS assay could complete the amplification process at 37°C constant temperature within 30 min and results could be visualized by the naked eye within 10 min on LFS. The assay displayed high sensitivity with a limit of detection of 3.05 CFU/reaction. It also demonstrated high specificity by showing no cross reaction with other pathogenic bacteria, and rapidness by being completed in less than an hour. Furthermore, when used with clinical samples, the assay had a coincidence rate of 98.26% with the culture-biochemical method and a kappa index value of 0.9433. These data indicate that the RPA-LFS assay represents a major improvement for P. aeruginosa detection, especially in resource-limited areas.

Purpose: This study aimed to investigate the prevalence of the CRISPR-Cas system in nosocomial isolates of Enterococcus and their possible association with antibiotic resistance and virulence genes. Materials and Methods: Identification and antimicrobial susceptibility of the microorganism were performed by the automatized VITEK 2 Compact system (bioMerieux, France). A total of 100 Enterococcus isolates were collected and identified by VITEK 2 Compact automatic microbial identification drug susceptibility analyzer. The prevalence of various CRISPR-Cas systems, antibiotic resistance genes and virulence genes were investigated by polymerase chain reaction (PCR). The prevalence of CRISPR-Cas systems associated with antibiotic resistance and virulence genes was performed by appropriate statistical tests. Results: A total of 100 isolates of Enterococcus were identified and there were 62/100(62.0%) Enterococcus faecalis isolates and 38/100(38.0%) Enterococcus faecalis isolates. In total, 46 (46.0%) of 100 isolates had at least one CRISPR-Cas locus. CRISPR elements were more prevalent in Enterococcus faecalis isolates. The results of PCR demonstrated that CRISPR1-Cas, orphan CRISPR2, and CRISPR3-Cas were present in 23 (23.0%), 42 (42.0%) and 5 (5.0%) Enterococcus isolates, respectively. Compared with CRISPR-Casnegative isolates, the CRISPR-Cas positive isolates showed significant lower resistance rates against ampicillin, erythromycin, levofloxacin, tetracycline, vancomycin, gentamicin, streptomycin, and rifampicin. Presumably consistent with drug susceptibility, fewer CRISPR loci were identified in vanA, tetM, ermB, aac6’-aph(2”), aadE, and ant(6) positive isolates. There was a significant negative correlation between the CRISPR-Cas locus and the enterococcal virulence factors enterococcal surface protein (esp) gene. Conclusion: In conclusion, the results indicated that the absence of the CRISPR-Cas system was negatively associated with some antibiotic resistance in clinical isolates of Enterococcus faecalis and Enterococcus faecium. Also, there was a negative correlation with the carriage of antibiotic resistance genes. Furthermore, CRISPR-Cas may prevent some isolates from acquiring certain virulence factors.