Multidrug efflux pumps function at the frontline to protect bacteria against antimicrobials by decreasing the intracellular concentration of drugs. This protective barrier consists of a series of transporter proteins, which are located in the bacterial cell membrane and periplasm and remove diverse extraneous substrates, including antimicrobials, organic solvents, toxic heavy metals, etc., from bacterial cells. This review systematically and comprehensively summarizes the functions of multiple efflux pumps families and discusses their potential applications. The biological functions of efflux pumps including their promotion of multidrug resistance, biofilm formation, quorum sensing, and survival and pathogenicity of bacteria are elucidated. The potential applications of efflux pump-related genes/proteins for the detection of antibiotic residues and antimicrobial resistance are also analyzed. Last but not least, efflux pump inhibitors, especially those of plant origin, are discussed.

Rational use of antibiotic is the key approach to improve the antibiotic performance and tackling of the antimicrobial resistance. The efficacy of antimicrobials are influenced by many factors: (1) bacterial status (susceptibility and resistance, tolerance, persistence, biofilm) and inoculum size; (2) antimicrobial concentrations (mutant selection window and sub-inhibitory concentration); (3) host factors (serum effect and impact on gut micro-biota). Additional understandings regarding the linkage between antimicrobial usages, bacterial status and host response offers us new insights and encourage the struggle for the designing of antimicrobial treatment regimens that reaching better clinical outcome and minimizing the emergence of resistance at the same time.

Food contamination with antibiotic residues has become a worldwide problem due to the heavy and improper use of antibiotics. The development of fast, high-throughput, easy, and economical screening methods for the detection of antibiotic residues is an important requirement as an alternative to the traditional assays. Immunoassays are widespread promising methods that can achieve the conditions of an analytical method in the evaluation of food and environmental protection. This review offers an overview of the preparation of antibodies and the currently established immune-assays for the antibiotic residues detection, including enzyme-linked immunosorbent assay, fluorescence immunoassay, radioimmunoassay, colloidal gold immunoassay, and chemiluminescence immunoassay. Furthermore, it will elaborate on the future perspective on the performance and improvement of these assays for the determination of antibiotic residues in food samples. These assays include a simple sample preparation and high-sensitive detection that offer a significant assurance for the screening of antibiotic residues. GRAPHICAL ABSTRACT

Public unrest about the use of antimicrobial agents in farming practice is the leading cause of increasing and the emergences of Multi-drug Resistant Bacteria that have placed pressure on the agri-food industry to act. The usage of antimicrobials in food and agriculture have direct or indirect effects on the development of Antimicrobial resistance (AMR) by bacteria associated with animals and plants which may enter the food chain through consumption of meat, fish, vegetables or some other food sources. In addition to antimicrobials, recent reports have shown that AMR is associated with tolerance to heavy metals existing naturally or used in agri-food production. Besides, biocides including disinfectants, antiseptics and preservatives which are widely used in farms and slaughter houses may also contribute in the development of AMR. Though the direct transmission of AMR from food-animals and related environment to human is still vague and debatable, the risk should not be neglected. Therefore, combined global efforts are necessary for the proper use of antimicrobials, heavy metals and biocides in agri-food production to control the development of AMR. These collective measures will preserve the effectiveness of existing antimicrobials for future generations.

Salmonella is recognized as one of the most common microbial pathogens worldwide. The bacterium contains the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, providing adaptive immunity against invading foreign nucleic acids. Previous studies suggested that certain bacteria employ the Cas proteins of CRISPR-Cas systems to target their own genes, which also alters the virulence during invasion of mammals. However, whether CRISPR-Cas systems in Salmonella have similar functions during bacterial invasion of host cells remains unknown. Here, we systematically analyzed the genes that are regulated by Cas3 in a type I-E CRISPR-Cas system and the virulence changes due to the deletion of cas3 in Salmonella enterica serovar Enteritidis. Compared to the cas3 gene wild-type (cas3 WT) Salmonella strain, cas3 deletion upregulated the lsrFGBE genes in lsr (luxS regulated) operon related to quorum sensing (QS) and downregulated biofilm-forming-related genes and Salmonella pathogenicity island 1 (SPI-1) genes related to the type three secretion system (T3SS). Consistently, the biofilm formation ability was downregulated in the cas3 deletion mutant (Δcas3). The bacterial invasive and intracellular capacity of Δcas3 to host cells was also reduced, thereby increasing the survival of infected host cells and live chickens. By the transcriptome-wide screen (RNA-Seq), we found that the cas3 gene impacts a series of genes related to QS, the flagellum, and SPI-1-T3SS system, thereby altering the virulence phenotypes. As QS SPI-1-T3SS and CRISPR-Cas systems are widely distributed in the bacteria kingdom, our findings extend our understanding of virulence regulation and pathogenicity in mammalian hosts for Salmonella and potentially other bacteria.

Due to the extensive application of antibiotics in medical and farming practices, the continued diversification and development of antimicrobial resistance (AMR) has attracted serious public concern. With the emergence of AMR and the failure to treat bacterial infections, it has led to an increased interest in searching for novel antibacterial substances such as natural antimicrobial substances, including microbial volatile compounds (MVCs), plant-derived compounds, and antimicrobial peptides. However, increasing observations have revealed that AMR is associated not only with the use of antibacterial substances but also with tolerance to heavy metals existing in nature and being used in agriculture practice. Additionally, bacteria respond to environmental stresses, e.g., nutrients, oxidative stress, envelope stress, by employing various adaptive strategies that contribute to the development of AMR and the survival of bacteria. Therefore, we need to elucidate thoroughly the factors and conditions affecting AMR to take comprehensive measures to control the development of AMR.