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Research progress on antibiotic resistance of Salmonella

Research progress on antibiotic resistance of Salmonella Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Yizhe Wang, Hengwei Ge, Xinyue Wei, Xihong Zhao* Research Center for Environmental Ecology and Engineering, Key Laboratory for Green Chemical Process of Ministry of Education, Hubei Key Laboratory of Novel Reactor and Green Chemical Technology, School of Environmental Ecology and Biological Engineering, Wuhan Institute of Technology, Wuhan 430205, China. *Corresponding author: Prof. Xihong Zhao, Ph. D School of Environmental Ecology and Biological Engineering, Wuhan Institute of Technology, Wuhan 430205, China. E-mail: xhzhao2006@gmail.com © The Author(s) 2022. Published by Oxford University Press on behalf of Zhejiang University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 ABSTRACT Antibiotic abuse results in various antibiotic resistance among a number of foodborne bacteria, posing a severe threat to food safety. Antibiotic resistance genes are commonly detected in foodborne pathogens, which has sparked much interest in finding solutions to these issues. Various strategies against these drug-resistant pathogens have been studied, including new antibiotics and phages. Recently, a powerful tool has been introduced in the fight against drug-resistant pathogens, namely, clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) system aggregated by a prokaryotic defense mechanism. This review summarized the mechanism of antibiotic resistance in Salmonella and resistance to common antibiotics, analysed the relationship between Salmonella CRISPR-Cas and antibiotic resistance, discussed the changes in antibiotic resistance on the structure and function of CRISPR-Cas, and finally predicted the mechanism of CRISPR-Cas intervention in Salmonella antibiotic resistance. In the future, CRISPR-Cas is expected to become an important tool to reduce the threat of antibiotic-resistant pathogens in food safety. Keywords: foodborne pathogens, antibiotic resistance, CRISPR-Cas, food safety Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 1. Introduction During food production, processing, storage, distribution, and preparation, foodborne pathogens are exposed to a variety of environmental factors, causing food poisoning or using food as a transmission medium. Food poisoning is caused by Salmonella enteritidis, and the toxin enters the host cell directly (Besser, 2018). The ability of Escherichia coli O157:H7 to produce a considerable amount of vero toxin was a distinguishing trait (Li et al., 2017a). Shigella has strong endotoxins and exotoxins(Ren et al., 2017). Staphylococcus aureus contains many enterotoxins, such as SEA, SEB, SEC and so on (Mehraj et al., 2016). Foodborne pathogens have become a serious food safety issue, causing diarrhea, dehydration, acute enteritis, shock, and even death. According to the World Health Organization (WHO) database, 41% of deaths related to diarrhea worldwide were attributed to Salmonella, which belongs to the Gram-negative intestinal bacilli (Majowicz et al., 2010; Besser 2018). According to the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas++ Web site (https://crisprcas.i2bc.paris-saclay.fr/MainDb/TaxoTree), it can be divided into Salmonella bongori, Salmonella enterica, unclassified Salmonella (Salmonella sp. HNK130, S13 and SCFS4), Salmonella sp. SSDFZ54 and Salmonella sp. SSDFZ69. Additionally, enteritidis caused by foodborne Salmonella has arisen for decades owing to food consumption. For example, infection with S. Typhimurium may cause typhoid-like symptoms and gastroenteritis with diarrhea in animals and humans, respectively (Pradhan et al., 2019). According to recent reports, a few drug-resistant hyperactive Salmonella strains may be screened from the host (Maka et al., 2016; McDermott et al., 2018; Casaux et al., 2019). Another study showed that the resistance rate of Salmonella typhimurium to ampicillin was Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 as high as 76.61% and high as that to another antibiotic (Liang et al., 2019). However, Salmonella has possessed resistant to polymyxin, which is the "last line of defense". Because mcr-1 was the resistance gene of polymyxin, it reduced the sensitivity of Salmonella (Liu et al., 2016). The CRISPR-Cas adaptive immune system controls gene transfer through conjugation and transformation, which enhances the transduction of plasmids, chromosomal genes and pathogenic islands (Watson et al., 2018). For Shigella, CRISPR is contributed to interfere with HGT and spontaneously regulates whether to acquire positive or negative genes (Chen et al., 2019). However, Salmonella possesses multiple antibiotic resistance, such as simultaneous resistance to chloramphenicol and streptomycin (Zhao et al., 2016). Therefore, it is critical to investigate the resistance of Salmonella. The CRISPR structure indicated that they were transcribed into small RNA (smRNA), which acted as the promoter and might play a role as small interfering RNA (siRNA) to prevent the entry of foreign sequences (Grissa et al., 2007). 87% of archaea and 45% of bacteria possess CRISPR structures (data from CRISPR Finder Program online, https://crispr.i2bc.paris-saclay.fr/), including spacers, direct repeats (DR), protospacer adjacent motif (PAM) and cas genes cluster. In particular, Salmonella has two types of CRISPR/Cas structures, which could be classified as types Ⅰ -E (Figure 1) based on the cse1 and cse2 genes (Makarova and Koonin, 2015). From Figure 1, Salmonella with CRISPR1 has a cas gene cluster structure, which shows that CRISPR1 has complete nucleic acid cleavage ability, and CRISPR2 is more seriously infected by phage. However, the computer language calculation of some websites showed that the CRISPR structure without cas gene cluster did not exhibit immune response function and was not a part of the real CRISPR structure in theory (Patterson et al., 2017). When a phage first infected, the surviving bacteria Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 incorporated portions of the phage's DNA into their spacers under the action of a nucleic acid polymerase expressed by the cas gene. In the next infection, based on the spacer as a template, cas gene expressed a nucleotide cutting enzyme, and an immune response occurred by shearing the exogenous genetic material (Figure 2). In addition, CRISPR played an important role in molecular biology based on cas12 and cas13, such as building a quick detection method for COVID-19 (Yan et al., 2019; Xiang et al., 2020). 2. Antibiotic resistance mechanism of Salmonella Currently, the way that bacteria acquire resistance genes is complex and diverse, for example, the selective pressure of antibiotics. However, evolution was beneficial for themselves, not humans, and would allow bacteria to be more environmentally adapted (Horn and Bhunia, 2018). Through a literature summary, resistance mechanisms of foodborne pathogens can be divided into the following four categories. First, plasmid-mediated resistance of Salmonella came from foreign plasmids. For example, oqxAB and aac(6’)Ib-cr on different plasmids combined with a single gyrA mutation in S. typhimurium may produce sufficient resistance to ciprofloxacin (CIP) (Wong et al., 2014), which may be realized by HGT. However, E. coli and Salmonella might improve their level of antibiotic tolerance by exchanging resistance genes through HGT (Oladeinde et al., 2019). Moreover, phage transduction is another drug resistance gene among foodborne pathogens, such as E. coli and Salmonella enteritidis (Gabashvili et al., 2020). However, plasmid-mediated production of the enzyme inactivated fluoroquinolones as well as amino acid glycosides and efflux pumps out the resistant drug, resulting in reduced antibiotic susceptibility but not to clinical levels unless chromosomal variations were present (Dolejska and Papagiannitsis, 2018; Jacoby, 2018). Additionally, plasmid-mediated colistin resistance Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 gene mcr-1 might generate colistin resistance, and plasmids were utilized as vehicles for horizontal gene transfer, which can be carried out by epidemic plasmids (inci2, incx4, inchi2, etc.) (Yi et al., 2017). Unfortunately, some research found a new resistance gene transfer by plasmids, mcr-2 loci on IncX4 plasmid, which showed 76.7% nucleotide identity to mcr-1 (Xavier et al., 2016). Second, the mutation or deletion of antibiotic binding sites of foodborne pathogens reduces the sensitivity of targeted antibiotics. The major targets of antibiotics in most foodborne pathogens are (i) the cell envelope, (ii) the ribosome, and (iii) nucleic acids (Foster, 2017). Aminoglycosides, tetracyclines, macrolides, and other drugs mainly bind to bacterial ribosomes to inhibit the synthesis of bacterial proteins, resulting in the resistance of bacteria to antibacterial drugs. In general, gyrA, gyrB, parC, and parE constitute the fluoroquinolone antibiotic resistance determining region, and mutation of gyrA leads to a change in the antibiotic binding site, resulting in the development of resistance to antibiotics (Wiuff et al., 2000). Under antibiotic pressure, surviving Salmonella produced the nucleotide base mutation, carrying genes of other Salmonella, which may be a homology relationship, and the resistance genes could escape from CRISPR structural immunity (Piddock, 2002). The DNA of Salmonella possessed single nucleotide polymorphism, which was able to produce resistance to any antibiotic. For example, the polymorphism of the TLR4 gene affected the recognition of Salmonella lipopolysaccharide by toll-like receptor 4, leading to positive or negative effects on human or animal resistance to Salmonella infection (Li et al., 2017b). However, it was difficult to determine whether the pathogenic bacteria were resistant at the beginning, and there would be some mutation sites that were not obvious. It was impossible to distinguish whether the drug resistance was caused by site Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 mutations or other reasons in clinical situations. A previous study reported the directed evolution with random genomic mutations, which could predefine the possibility of mutations in the whole genome sequence of pathogenic bacteria with a million-fold mutation rate, provide a rapid identification of bacterial drug resistance research with the help of this research, and distinguish single drug-resistant bacteria from multidrug-resistant bacteria (Nyerges et al., 2018). The mutant drug resistance gene might cover a wide range of different pathogenic bacteria along with the transmission of the plasmid. Third, the active efflux pump of Salmonella is mainly AcrAB-TolC of the resistance- nodulation-division family. Overexpression of efflux pump regulated genes would lead to drug resistance, recognizes multiple substrates and is associated with multidrug resistance (MDR) (Blair et al., 2014). AcrAB-TolC is composed of periplasmic protein AcrA, efflux transporter AcrB and outer membrane channel protein TolC, which are able to discharge antibiotics outside the cell through the central channel (Tikhonova and Zgurskaya, 2004). After the induction of a Salmonella ciprofloxacin highly resistant strain (resistance value 256 μg/mL) through inactivation of AcrB, the resistance value dropped to 0.25 μg/mL (Siew et al., 2007). What is more, RamA is an AraC/XylS transcription activator and homologue of MarA and regulates the expression of genes encoding the AcrAB-TolC MDR efflux pump (Abouzeed et al., 2008). Similar to E. coli and other Enterobacteriaceae (Figure 3), ramA also regulates the expression of acrAB and tolC (Ricci and Piddock, 2009). In addition, point mutations in ramR, which is the repressor of ramA transcription, as well as ramA and acrB overexpression have all been associated with multidrug resistance (Abouzeed et al., 2008; Ricci and Piddock, 2009). Another study has confirmed that the separation of MarR from the promoter region may promote the expression of MarA, and the MarA protein could activate Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 acrAB and micF and trigger Salmonella typhimurium MDR (Alekshun and Levy, 1997; Prouty and A., 2004). Telke et al. (2017) found that the expression of soxRS may activate the upregulation of acrAB, contributing to the resistance to colistin. In addition, there were distinctions in efflux pump regulatory genes among Bacteroidetes, such as Klebsiella pneumoniae, romA, and rarA, which could regulate acrAB (Rosenblum et al., 2011). Therefore, as an important resistance mechanism, the acrAB pump needs further investigation to solve the problem of antibiotic resistance in Salmonella. A platform based on efflux pump gene expression may be a promising rapid detection method for foodborne pathogens. Finally, there were changes in the bio-membrane permeability. Generally, drugs entering pathogenic bacteria require passage through outer membrane porin (OMP) on the cell membrane (Masi et al., 2019). However, the abnormality in OMP would cause the permeability of bacterial cell membrane to affect the drug resistance of pathogenic bacteria (Hu et al., 2011). Compared to the resistant and sensitive strains of ciprofloxacin, the expression of outer membrane porins F (OmpF) in the resistant strains decreased significantly (Du et al., 2020). Interestingly, OmpF effectively controlled the entry and exit of antibiotics and only allowed antibiotics to enter slowly, which improved the drug resistance rate (Delcour 2009). The loss of OmpC protein led to carbapenem resistance, while OmpC , c d OmpF , and Omp proteins determined the resistance mechanism of Salmonella membrane porin (Vergalli et al., 2020). However, OMP did not play a separate role in the mechanism of drug resistance. Indeed, it regulated the expression levels of ompD and acrD and controlled the penetration of antibiotics to produced drug resistance (Hu et al., 2009). Particularly, OmpF, a Salmonella drug channel, was regulated by MarA and SoxS proteins. Specifically, Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 the expression of marA or soxS activated the transcription of MicF (antisense RNA of OmpF RNA) and prevented the translation of OmpF mRNA, which improved the drug resistance rate by the degradation of OmpF RNA (Hartog et al., 2008). A previous research demonstrated that an OMP-based typing method quickly detected the drug resistance of Salmonella and the expression of OMP protein (Kumar et al., 2019). In general, the above four modes constituted the antibiotic resistance mechanism of Salmonella. The increase in antibiotic dose changed the antibiotic resistance of Salmonella, resulting in resistance gene mutation, which limited the effect of antibiotics. What is more, molecular biology is also committed to exploring better mitigation measures. Therefore, the investigation of antibiotic resistance genes and CRISPR-Cas mechanism may solve these problems. 3. Salmonella resistance to different antibiotics 3.1. Resistance to quinolones and fluoroquinolones Salmonella showed resistance to fluoroquinolones. For example, the fluoroquinolones resistance determining region was composed of gyrA, gyrB, parC, and parE, which played a role in antibiotic resistance by inhibiting the gyrase of Gram-negative bacteria and topoisomerase IV of Gram-positive bacteria (Akiba et al., 2007). Another study demonstrated that the ser83 subunit of Salmonella gyrA site was mutated to phenylalanine and then showed resistance to naphthyridic acid (MIC 256 µg/mL) and ciprofloxacin (MIC 0.25-2 µg/mL); similarly, the asp87 site was mutated into glycine and tyrosine (MIC 256- 512µg/mL of naphthyridic acid and MIC 0.12-0.5 µg/mL of ciprofloxacin) (Chen et al., 2007; Fàbrega et al., 2008). Some researchers suggested that the resistance of Salmonella to naphthyridic acid, enrofloxacin, ciprofloxacin, ofloxacin and levofloxacin and the proportion Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 of antibiotic-resistant strains were 90.2%, 39.0%, 41.5%, 29.3%, and 26.8%, respectively (Fàbrega et al., 2008). Antibiotic resistance in Salmonella should not be underestimated. In addition, researchers have identified a new gene regulatory region of Salmonella resistance to antibiotics called plasmid mediated quinolone resistance (PMQR) (Martínez- Martínez et al., 1998). Moreover, a multi-antibiotic resistant plasmid from clinical isolates of Klebsiella pneumoniae was able to increase Salmonella resistance to fluoroquinolones. The multi-antibiotic plasmid consisted of qnrA, qnrB, qnrC, qnrD and qnrS (Nordmann, 2006), and each family contains different subunits and mutants, such as QnrB1, QnrB7, and QnrB17 (Jacoby et al., 2008). However, Salmonella and Escherichia coli isolated from human feces carried plasmids with qnrA, qnrB, and qnrS resistance genes, which showed resistance to fluoroquinolones (Doma et al., 2020). Apart from PMQR, another resistance mechanism of quinolones and fluoroquinolones was the production of a modified aminoglycoside acetyltransferase (AAC(6)-b-cr), which reduced the activity of the compound through enzyme modification and the activity of ciprofloxacin by n-acetylation of the amino nitrogen on the piperazinyl substituent (Robicsek et al., 2006). Salmonella plays an essential role in antibiotic resistance through AcrAB-TolC efflux system. Depending on the expression of acrAB, the damage of eight transporters would result in the construction of multigene deletion mutants (ΔacrA, ΔacrB, ΔtolC, ΔacrAB, ΔacrEF, ΔacrD, ΔmdsABC, ΔmdtABC, ΔemrAB, ΔmacAB, ΔmdfA, ΔmdtK, ΔacrAB-ramA, ΔacrAB-marA, and ΔacrAB-soxS), and ramA and marA contributed to coordinating the regulation feedback mechanism of AcrAB-Tolc and 8 other functional efflux pumps (Zhang et al., 2018). Additionally, Salmonella isolated from the environment generally gained PMQR genes (59%), including qnrS1, aac(6)-1b-cr, qnrB19, and qnrD genes (Veldman et al., 2011). Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 In addition to the gyrA mutation in the fluoroquinolone determining region (Yang et al., 2012), a common drug resistance gene oqxAB based on chloramphenicol and fluoroquinolone antibiotics was also found in Hong Kong, China (Wong and Chen, 2013). 3.2. Resistance to sulfonamides and trimethoprim Sulfamethoxazole (SMZ) is a synthetic antibacterial drug that has been used clinically for nearly 50 years. The combined application of trimethoprim (TMP) and SMZ enhanced the antibacterial effect and expanded the treatment range (Falagas et al., 2015; Vila-Costa et al., 2017). However, after long-term use, broad-spectrum antibiotics seemed to become less effective. SMZ is usually associated with mutations in the gene encoding dihydrofolate synthase (DHPS). However, clinical antibiotic-resistant mutants possessed additional compensatory mutations in DHPS so that the antibiotic-resistant system worked normally (Sköld 2000). The resistance of Salmonella to sulfonamides drugs was related to the presence of the sul (sul1, sul2 and sul3) genes, which was the encoding forms of DHPS that were not inhibited by antibiotics (Antunes et al., 2005). Moreover, the mechanism of Salmonella resistance to sulfonamides was mainly plasmid transduction. For example, sul1 was lied in 3’-conserved segment (3’-CS) of class 1 integrons (Sköld 2000). However, sul2 exists on antibiotic- resistant plasmids, and Salmonella might exhibit resistance by controlling the expression of plasmids (Enne et al., 2004). The drug resistance gene sul3 came from a wide range of sources and has been detected in large plasmids of different Salmonella (Guerra et al. 2004). Similar to SMZ, trimethoprim also competed with essential folate pathway substrates in bacteria and inhibited dihydrofolate reductase and dihydrofolate reductase variants, which reduced the sensitivity of Salmonella to trimethoprim (Korsak & Krawczyk-Balska 2017). Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Researchers reported mutants of dhfr Ⅰ and dhfr Ⅱ in Gram-negative intestinal bacteria, which made the MIC of TMP 11000 times higher than that of normal (Huovinen 2001). At present, researchers often detect the Sul gene family of Salmonella in meat products (Xu et al., 2020). In research, 58.1% of Salmonella were resistant to SMZ, and 34% were resistant to TMP (Thai et al., 2012b). Among the meat sold in Canada, 110 strains of Salmonella were identified, 71% of which were resistant to SMZ, and sul1, sul2, and sul3 resistance genes were detected in the isolated Salmonella (Aslam et al., 2012). 3.3. Resistance to β-lactams β-Lactams are widely used in foodborne pathogens and have high antibacterial activity. The invention of lactam drugs alleviated the negative reaction of human beings to microbial infection and the embarrassing situation of dealing with pathogens without available antibiotics. The abuse of antibiotics brought about the emergence of extended spectrum β- lactamases (ESBLs) resistant strains. ESBLs hydrolyzed penicillin and cephalosporins (excluding cephalosporins). However, this drug resistance mechanism is not effective for a long time. The hydrolysis efficiency of ESBLs was inversely proportional to that of β-lactam inhibitors, currently known related drug resistance genes include , , , and (Winokur et al., 2000). Resistance genes such as bla (detection rate CTX-M 94.1%), (45%), and (24.1%) were detected in isolates (Kuang et al., 2018). In addition, the monitoring of pAmpC-harboring and ESBL-producing Salmonella has also become particularly important. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 3.4. Resistance to chloramphenicol Chloramphenicol acetyltransferases (CATs) regulated the antibiotic resistance of chloramphenicol resistance in foodborne pathogenic bacteria. Affected by HGT, the cat gene is widely expressed in foodborne pathogenic bacteria. The cat gene of Salmonella was expressed from plasmids of other pathogenic bacteria or mutation, and the cat gene family also contains the cata1, cata2, and variant catB genes (Villa et al., 2002; Chen et al., 2004; Ahmed et al., 2005). The production of new mutations in Salmonella also improved the sensitivity to chloramphenicol. In addition, cmlA and floR effectively inhibited the expression of efflux pump genes, prevented the outward penetration of chloramphenicol and reduced the sensitivity of chloramphenicol (Thai et al., 2012a). Salmonella isolated from meat products contained at least one antibiotic resistance gene, of which 57.1% Salmonella contained floR, 50% contained cmlA1, and 14.3% contained dual antibiotic resistance genes (Thai et al., 2012a). A food sampling inspection in Germany showed that the detection rate of floR gene in antibiotic-resistant Salmonella was the highest (90.9%), while the detection rates of catA and cmlA were lower (Miko et al., 2005). Similarly, the detection frequency of cat1 and cat2 in antibiotic-resistant Salmonella was very low, which explained that they were not the main chloramphenicol resistance regulatory genes (Thong and Modarressi, 2011). As described, antibiotic resistance gene mediation was the main reason why Salmonella was sensitive to chloramphenicol. The above research proved that floR gene may be transferred in Salmonella through HGT, and the HGT frequency of cat1 and cat2 genes was not very high. At present, the main factors of Salmonella resistance come from HGT of plasmids. In addition, the interaction between drug resistance genes and CRISPR immune mechanism needs to be further studied. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 3.5. Brief summary of resistance The mechanism of drug resistance was generally described, and the resistance genes were summarized, which laid a foundation for the follow-up related resistance research. Notably, some other broad-spectrum antibiotic resistance gene loci were statistically analysed (Table 1) (Karczmarczyk et al., 2010; Deekshit et al., 2012). From Table 1, gyrA, gyrB, parC, and parE are the determinants of drug resistance to fluoroquinolones. To date, five different qnr families, each with a different number of alleles (qnrA1-7, qnrS1-4, qnrB1-31, qnrC and qnrD) and oqxAB, have also been widely studied (Table 1). For example, the qnr family is common in poultry, pigs, sheep and turkeys, especially qnrS1 in E. coli isolated from humans, and 59% of Salmonella contains the qnr family (Veldman et al., 2011). Research on the resistance genes of Salmonella to sulfonamides showed that 152 strains of sul1 gene (76%), 74 strains of sul2 gene (37%) and 14 strains of sul3 gene (7%) were detected in 200 resistant strains, and sul1 and sul2 emerged in 24 isolates at the same time (Antunes et al., 2005). However, 34% of foodborne Salmonella were highly resistant to trimethoprim (Thai et al., 2012a). The trimethoprim resistance genes of Salmonella are shown in Table 1, and Deekshit found that Salmonella Weltevreden contained a class 1 integron with a single gene dfrA7 in the integron cassette and Salmonella Newport with dhfrA1(Deekshit et al., 2012). In fact, the study found that there are many kinds of β-lactam antibiotic resistance genes of Salmonella (Table 1). The majority of cephalosporin resistant Salmonella express a broad-spectrum β-lactamase, which can hydrolyze oximine cephalosporins but not cephalosporins (Winokur et al., 2000). In addition, bla , bla , bla and bla β-lactamase genes were validated by PCR, and CMY ctx-m TEM OXA Salmonella containing plasmid-mediated bla gene increased from 0% (0/27) in 2000 and CMY-2 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 2001 to 75% in 2004 and 2005, which especially had a higher frequency of infection than human with non-bla (Zaidi et al., 2007). The determinants of chloramphenicol CMY-2 resistance in Salmonella include catA1, floR and cmlA1. Although compared with the three antibiotics, the resistance rate of chloramphenicol is limited, and the induction effect on Salmonella resistance and antibiotic membrane activity is weak (Lorenzo 2019). 4. Study on CRISPR-Cas and antibiotic resistance of Salmonella Typically, cas genes are used to express DNA cleavage enzymes, combined with PAM localization, and spacers are used as templates to exert immune effects. Strains without CRISPR-Cas are more prone to HGT of antibiotic-resistant plasmids, which might be related to the immune mechanism of CRISPR-Cas (Palmer and Gilmore 2010). For example, Enterococcus faecalis obtained antibiotic resistance genes (AGRs) by escaping CRISPR-Cas immunity. For this reason, strains without CRISPR loci were more likely to obtain antibiotic resistance genes than strains with complete CRISPR loci. Salmonella contains complete CRISPR-Cas, and two antibiotic resistance genes, bla CMY-2 and aac(6’)-Iaa, were obtained from the incll plasmid (Monte et al., 2021). In addition, phages slowly helped Salmonella cope with the threat of antibiotics due to the transduction of antibiotic-resistant plasmids. The reason for the decreased sensitivity of Salmonella to ciprofloxacin was that phage DT104 carried ARGs. The abovementioned phenomenon was explained by Salmonella apparently receiving ARGs and appearing in CRISPR-Cas as a new spacer, resulting in the emergence of antibiotic resistance (Threlfall et al., 2000). CRISPR-Cas typing was positively correlated with antibiotic resistance corresponding to serotypes in serotype verification (Hoelzer et al., 2010). At present, experiments show that the pressure of wild strains on antibiotics decreased and the sensitivity of antibiotics Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 increased. The clinical isolated Salmonella showed that the CRISPR-Cas structure had significant resistance to broad-spectrum antibiotics (DiMarzio et al., 2013). CRISPR-Cas usually coexists with antibiotic resistance genes in Salmonella. CRISPR-Cas not only obtained antibiotic resistance genes as new spacers but also adopted other genes beneficial to itself (Shehreen et al., 2019). One study showed that self-targeting spacers accounted for 0.4% of all spacers (including the vast majority of spacers without assignable original spacers) and appeared in 18% of the contained prokaryotic genome (Stern et al., 2010). The above study showed that the spacers abandoned the old sequences and adopted the new sequences, and there was the possibility of adding dissociative drug resistance genes to spacers. Studies have shown that Cas9 protein had enzymatic digestion of free exogenous RNA, even without PAM (O'Connell et al., 2014; Rousseau et al., 2018). Therefore, CRISPR-Cas of Salmonella also acted on the RNA products of resistance gene expression. In particular, Streptococcus thermophilus CRISPR1-Cas also obtained naturally antibiotic resistance genes of plasmids (Garneau et al., 2010). In addition, the spacers of Klebsiella pneumoniae found to be β- lactams, quinolones, and aminoglycosides were partially resistant fragments (Wang et al., 2020). With the emergence of resistance gene fragments in the spacers, perhaps the older spacers were removed, but the drug resistance of bacteria was not affected and was still in the drug resistance stage. Under the pressure of antibiotic selection, the CRISPR-Cas of Salmonella may be mutated, allowing the bacteria to obtain foreign resistance genes and survive (Li et al., 2021). The spacers mutation made the free resistant fragments unaffected by CRISPR-Cas. For example, E. coli retained the same spacers as the foreign plasmids, indicating that they had ancient contact with the plasmids, although spacers expression was no longer active (Touchon et al., 2012). The resistance gene fragments in the spacers would mutate autonomously under environmental pressure and weaken the intervention of Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 CRISPR-Cas on HGT. CRISPR-Cas in mammalian intestinal flora prevented the transmission of antibiotic-resistant plasmids, such as Enterococcus faecalis (Price et al., 2019a). On the other hand, type I-E of Klebsiella pneumoniae might interfere with the acquisition of plasmids containing antibiotic resistance genes to keep the strain sensitive to antibiotics (Li et al., 2018). Compared with CRISPR-Cas of Klebsiella pneumoniae, Salmonella was also type Ⅰ -E with the same antibiotic sensitivity mechanism. No plasmids or phage-targeted spacers were identified to match any drug resistance genes, so most of the drug resistance gene fragments in the spacers came from free fragments. Although anti-CRISPRs had evolved to provide CRISPR-Cas advantages to virulent and temperate phages, they evolved in a variety of ways (Landsberger et al., 2018). Anti-CRISPR benefited ARGs while reducing the defense ability of CRISPR-Cas against phages. Under antibiotic selection, Salmonella inserted the spacers into CRISPR-Cas to complete the immune response. Therefore, ARGs were obtained only in cells without mobile genetic elements targeting spacers, which was the reason why CRISPR-Cas targeted exogenous DNA, not ARGs (Shehreen et al., 2019). CRISPR-Cas and ARGs of bacteria could not exist at the same time because ARGs were incorporated into the spacers of CRISPR-Cas. In summary, ARGs might also be recognized and degraded by Cas protein due to bacterial immunity. Given the above, based on the theory that ARGs and CRISPR-Cas cannot co-exist, research introduced the use of CRISPR-Cas to solve the problem of bacterial antibiotic resistance (Aslam et al., 2020). Moreover, CRISPR was more commonly used to analyse Salmonella serotypes with advantages compared with pulsed field gel electrophoresis and multi-locus sequence analysis (DiMarzio et al., 2013; Vosik et al., 2018). Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 CRISPR-Cas9 gene editing technology was the most important discovery in this century. It showed that mature crRNA formed a special double-stranded RNA structure with tracr- RNA through base complementary pairing, which guided Cas9 protein to cause double- strand breaks in the target DNA (Gupta et al. 2019). CRISPR-Cas9 removed the plasmids carrying mcr-1 in intestinal pathogens and improved the sensitivity of intestinal bacteria to colistin (Dong et al., 2019). After inserting artificially edited Cas9, which acted on the virulence genes of Staphylococcus aureus, and destroyed the plasmids carrying antibiotic resistance genes (Bikard et al., 2014). Especially, CRISPR-Cas9 antimicrobials function in vivo to kill S. aureus in a mouse skin colonization model. In addition, the artificially edited CRISPR-Cas9 purposefully acted on the elimination of antibiotic-resistant plasmids and phage invasion while protecting antibiotic-sensitive bacteria (Yosef et al., 2015). This strategy could be applied to the fermentation of hospital hand sanitizer and engineered bacteria. Combined with previous research, it is speculated that the coupling mechanism is the best way to provide CRISPR-Cas9 antibacterial agent to bacteria (Dong et al., 2019). Based on the above theory, the inhibition of the HGT effect by artificial CRISPR-Cas9 transduction increased the sensitivity of Salmonella to antibiotics. The crRNA and tracr-RNA synthesize polymer small guide RNA (sgRNA), which was designed to eliminate antibiotic resistance genes from bacterial DNA (Gomaa et al., 2014). Moreover, CRISPR-Cas9 reduced the occurrence of antibiotic resistance in a sequence-specific manner (Rodrigues et al., 2019). For instance, Campylobacter lacks cas9, which improves its sensitivity to antibiotics and the level of HGT (Shabbir et al., 2018). Cas9 deletion increased the antibiotic sensitivity of C. jejuni, which may be related to cas9 preventing the HGT of resistance genes. Given the above, plasmids or phages carryingCRISPR-Cas9 system with specific sequences can selectively remove drug-resistant bacteria from complex strains (Bikard et al., 2014). As Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 described, based on drugresistant Salmonella isolated from clinical or poultry feed, it is possible to eliminate drug-resistant genes by CRISPR-Cas9 plasmids, and even the plasmids contain multiple resistance gene templates. If the restriction of PAM codon is overcome, it will also be possible to eliminate multiple resistance genes at one time. Moreover, similar to the construction of E. coli movable CRISPR-Cas genetic elements (Yosef et al., 2015), cas gene clusters were combined with lysogenic bacteriophages. The cse1, cse2, cas7, cas5, cas6 and cas3 contained in Salmonella CRISPR-Cas were enough to knock out the target resistance genes. A series of pIS26-CRISPR/Cas9 suicide plasmids were constructed, and specific guiding RNAs targeting Incx4, Inci2 and Inchi2 plasmid replication genes and drug resistance genes mcr-1, blakpc-2 and blandm-5 were designed, and the plasmid of CRISPR- Cas9 based on IS26 can be effectively used as an immune system to prevent the acquisition of exogenous resistant plasmid again (He et al., 2021). Therefore, it is feasible to mutate the drug resistance gene fragments of CRISPR-Cas9 plasmids or phages, and it has a better treatment effect on the drug resistance of pathogenic bacteria. At present, the application of plasmid or phage containing CRISPR-Cas9 in Salmonella is limited, and a lot of studies need to be carried out. In particular, Price et al. (2019b) found that CRISPR-Cas effectively prevented the HGT effect of antibiotic resistance genes in mammalian intestinal flora. With the continuous exploration of CRISPR-Cas, more strategies may emerge to inhibit food safety problems caused by foodborne pathogens. In addition, CRISPR-Cas is not only directly related to drug resistance but also slightly related to quorum sensing (QS) of pathogenic bacteria. For example, there has also been some progress in researches on the relationship between QS and CRISPR. For instance, research showed that when the cas3 gene was missing in the CRISPR structure of Salmonella, the lsrFGBE genes related to QS were upregulated, and the biofilm forming ability decreased (Cui et al., 2020). Moreover, the Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 absence of AI-2 affects the immune acquisition of spacers in Salmonella CRISPR structure. QS pro-interfering enzyme in Pseudomonas aeruginosa significantly changed the expression of CRISPR-Cas genes (Mion et al., 2019). 5. Conclusions The food safety problem caused by Salmonella is becoming increasingly serious. The independent CRISPR-Cas structures among strains were significantly different in response to the HGT of pathogenic bacteria. HGT is the main transmission mechanism of drug resistance genes. The analysis showed that it was easier to obtain the resistant fragments without CRISPR locus, such as Enterococcus faecalis. It can be concluded that resistant genes in spacers mainly come from the free fragments and phages in the environment. Therefore, the spacers in CRISPR-Cas were different between drug-resistant and nondrug-resistant strains. In general, there was a close relationship between CRISPR-Cas and drug resistance in Salmonella, which was speculated to interfere with the expression of drug-resistant plasmids and cytoplasmic free resistant fragments. In addition, gene editing based on CRISPR-Cas9 accurately mutates the resistance genes in drug-resistant Salmonella. The review lays the foundation of food safety at the molecular level and provides a new research idea to solve the antibiotic resistance of foodborne pathogens. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Conflict of interest There are no conflicts to declare. Funding This study was supported by the Graduate Innovative Fund of Wuhan Institute of Technology (No. CX2020347) and the Key projects of science and technology research plan of Hubei Provincial Department of Education (D202015001), China. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 References Abouzeed, Y. M., Baucheron, S., Cloeckaert, A. (2008). ramR mutations involved in efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium. Antimicrobial agents and chemotherapy, 52(7): 2428-2434. Ahmed, A. 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(2016). Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, June 2016. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin, 21(27). Xiang, X., Qian, K., Zhang, Z., et al. (2020). CRISPR-cas systems based molecular diagnostic tool for infectious diseases and emerging 2019 novel coronavirus (COVID-19) pneumonia. Journal of drug targeting, 28(7-8): 727-731. Xu, Z., Wang, M., Zhou, C., et al. (2020). Prevalence and antimicrobial resistance of retail-meat-borne Salmonella in southern China during the years 2009-2016: The diversity of contamination and the resistance evolution of multidrug-resistant isolates. International journal of food microbiology, 333: Yan, F., Wang, W., Zhang, J. (2019). CRISPR-Cas12 and Cas13: the lesser known siblings of CRISPR- Cas9. Cell biology and toxicology, 35(6): 489-492. Yang, B., Xi, M., Cui, S., et al. (2012). Mutations in gyrase and topoisomerase genes associated with fluoroquinolone resistance in Salmonella serovars from retail meats. Food Research International, 45(2): 935-939. Yi, L. X., Liu, Y. Y., Wu, R., et al. (2017). Research progress on the plasmid-mediated colistin resistance gene mcr-1. Yi chuan = Hereditas, 392(): 110-126. Yosef, I., Manor, M., Kiro, R. et al. (2015). Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. Proceedings of the National Academy of Sciences of the United States of America, 112(23): 7267-7272. Zaidi, M. B., Leon, V., Canche, C., et al. (2007). Rapid and widespread dissemination of multidrug- resistant blaCMY-2 Salmonella Typhimurium in Mexico. The Journal of antimicrobial chemotherapy, 60(2): 398-401. Zhang, C. Z., Chen, P. X., Yang, L., et al. (2018). Coordinated Expression of acrAB-tolC and Eight Other Functional Efflux Pumps Through Activating ramA and marA in Salmonella enterica serovar Typhimurium. Microbial drug resistance (Larchmont, NY), 24(2): 120-125. Zhao, J., Zhang, Y., Xie, Z., et al. (2016). Characteristics of drug resistance and molecular typing research for Salmonella Enteritidis isolated in Henan province from 2011 to 2013. Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine], 50(3): 261-265. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Table 1 Examples of drug-resistant Salmonella isolated from food Type of antibiotic Resistance gene Reference Quinolones gyrA, gyrB, parC, parE Veldman K.et al and Karczmarczyk and qnrB, qnrD, qnrS, oqxAB M.et al Fluoroquinolones Sulfonamides sul1, sul2, sul3 Antunes P.et al and Guerra B.et al dfrA1, dfrA12, dfrV, dhfr1, dhfrV, Thai TH Chen S and Deekshit VK.et Trimethoprim dhfrA7, dhfr12, dhfr13, dhfr17 al , , , β-lactams , , , Aslam M and Chen S.et al Chloramphenicol catA1, floR, cmlA1 Miko A and Deekshit VK.et al Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Accepted Manuscript http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Food Quality and Safety Oxford University Press

Research progress on antibiotic resistance of Salmonella

Food Quality and Safety , Volume Advance Article – May 12, 2022

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Abstract

Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Yizhe Wang, Hengwei Ge, Xinyue Wei, Xihong Zhao* Research Center for Environmental Ecology and Engineering, Key Laboratory for Green Chemical Process of Ministry of Education, Hubei Key Laboratory of Novel Reactor and Green Chemical Technology, School of Environmental Ecology and Biological Engineering, Wuhan Institute of Technology, Wuhan 430205, China. *Corresponding author: Prof. Xihong Zhao, Ph. D School of Environmental Ecology and Biological Engineering, Wuhan Institute of Technology, Wuhan 430205, China. E-mail: xhzhao2006@gmail.com © The Author(s) 2022. Published by Oxford University Press on behalf of Zhejiang University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 ABSTRACT Antibiotic abuse results in various antibiotic resistance among a number of foodborne bacteria, posing a severe threat to food safety. Antibiotic resistance genes are commonly detected in foodborne pathogens, which has sparked much interest in finding solutions to these issues. Various strategies against these drug-resistant pathogens have been studied, including new antibiotics and phages. Recently, a powerful tool has been introduced in the fight against drug-resistant pathogens, namely, clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) system aggregated by a prokaryotic defense mechanism. This review summarized the mechanism of antibiotic resistance in Salmonella and resistance to common antibiotics, analysed the relationship between Salmonella CRISPR-Cas and antibiotic resistance, discussed the changes in antibiotic resistance on the structure and function of CRISPR-Cas, and finally predicted the mechanism of CRISPR-Cas intervention in Salmonella antibiotic resistance. In the future, CRISPR-Cas is expected to become an important tool to reduce the threat of antibiotic-resistant pathogens in food safety. Keywords: foodborne pathogens, antibiotic resistance, CRISPR-Cas, food safety Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 1. Introduction During food production, processing, storage, distribution, and preparation, foodborne pathogens are exposed to a variety of environmental factors, causing food poisoning or using food as a transmission medium. Food poisoning is caused by Salmonella enteritidis, and the toxin enters the host cell directly (Besser, 2018). The ability of Escherichia coli O157:H7 to produce a considerable amount of vero toxin was a distinguishing trait (Li et al., 2017a). Shigella has strong endotoxins and exotoxins(Ren et al., 2017). Staphylococcus aureus contains many enterotoxins, such as SEA, SEB, SEC and so on (Mehraj et al., 2016). Foodborne pathogens have become a serious food safety issue, causing diarrhea, dehydration, acute enteritis, shock, and even death. According to the World Health Organization (WHO) database, 41% of deaths related to diarrhea worldwide were attributed to Salmonella, which belongs to the Gram-negative intestinal bacilli (Majowicz et al., 2010; Besser 2018). According to the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas++ Web site (https://crisprcas.i2bc.paris-saclay.fr/MainDb/TaxoTree), it can be divided into Salmonella bongori, Salmonella enterica, unclassified Salmonella (Salmonella sp. HNK130, S13 and SCFS4), Salmonella sp. SSDFZ54 and Salmonella sp. SSDFZ69. Additionally, enteritidis caused by foodborne Salmonella has arisen for decades owing to food consumption. For example, infection with S. Typhimurium may cause typhoid-like symptoms and gastroenteritis with diarrhea in animals and humans, respectively (Pradhan et al., 2019). According to recent reports, a few drug-resistant hyperactive Salmonella strains may be screened from the host (Maka et al., 2016; McDermott et al., 2018; Casaux et al., 2019). Another study showed that the resistance rate of Salmonella typhimurium to ampicillin was Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 as high as 76.61% and high as that to another antibiotic (Liang et al., 2019). However, Salmonella has possessed resistant to polymyxin, which is the "last line of defense". Because mcr-1 was the resistance gene of polymyxin, it reduced the sensitivity of Salmonella (Liu et al., 2016). The CRISPR-Cas adaptive immune system controls gene transfer through conjugation and transformation, which enhances the transduction of plasmids, chromosomal genes and pathogenic islands (Watson et al., 2018). For Shigella, CRISPR is contributed to interfere with HGT and spontaneously regulates whether to acquire positive or negative genes (Chen et al., 2019). However, Salmonella possesses multiple antibiotic resistance, such as simultaneous resistance to chloramphenicol and streptomycin (Zhao et al., 2016). Therefore, it is critical to investigate the resistance of Salmonella. The CRISPR structure indicated that they were transcribed into small RNA (smRNA), which acted as the promoter and might play a role as small interfering RNA (siRNA) to prevent the entry of foreign sequences (Grissa et al., 2007). 87% of archaea and 45% of bacteria possess CRISPR structures (data from CRISPR Finder Program online, https://crispr.i2bc.paris-saclay.fr/), including spacers, direct repeats (DR), protospacer adjacent motif (PAM) and cas genes cluster. In particular, Salmonella has two types of CRISPR/Cas structures, which could be classified as types Ⅰ -E (Figure 1) based on the cse1 and cse2 genes (Makarova and Koonin, 2015). From Figure 1, Salmonella with CRISPR1 has a cas gene cluster structure, which shows that CRISPR1 has complete nucleic acid cleavage ability, and CRISPR2 is more seriously infected by phage. However, the computer language calculation of some websites showed that the CRISPR structure without cas gene cluster did not exhibit immune response function and was not a part of the real CRISPR structure in theory (Patterson et al., 2017). When a phage first infected, the surviving bacteria Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 incorporated portions of the phage's DNA into their spacers under the action of a nucleic acid polymerase expressed by the cas gene. In the next infection, based on the spacer as a template, cas gene expressed a nucleotide cutting enzyme, and an immune response occurred by shearing the exogenous genetic material (Figure 2). In addition, CRISPR played an important role in molecular biology based on cas12 and cas13, such as building a quick detection method for COVID-19 (Yan et al., 2019; Xiang et al., 2020). 2. Antibiotic resistance mechanism of Salmonella Currently, the way that bacteria acquire resistance genes is complex and diverse, for example, the selective pressure of antibiotics. However, evolution was beneficial for themselves, not humans, and would allow bacteria to be more environmentally adapted (Horn and Bhunia, 2018). Through a literature summary, resistance mechanisms of foodborne pathogens can be divided into the following four categories. First, plasmid-mediated resistance of Salmonella came from foreign plasmids. For example, oqxAB and aac(6’)Ib-cr on different plasmids combined with a single gyrA mutation in S. typhimurium may produce sufficient resistance to ciprofloxacin (CIP) (Wong et al., 2014), which may be realized by HGT. However, E. coli and Salmonella might improve their level of antibiotic tolerance by exchanging resistance genes through HGT (Oladeinde et al., 2019). Moreover, phage transduction is another drug resistance gene among foodborne pathogens, such as E. coli and Salmonella enteritidis (Gabashvili et al., 2020). However, plasmid-mediated production of the enzyme inactivated fluoroquinolones as well as amino acid glycosides and efflux pumps out the resistant drug, resulting in reduced antibiotic susceptibility but not to clinical levels unless chromosomal variations were present (Dolejska and Papagiannitsis, 2018; Jacoby, 2018). Additionally, plasmid-mediated colistin resistance Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 gene mcr-1 might generate colistin resistance, and plasmids were utilized as vehicles for horizontal gene transfer, which can be carried out by epidemic plasmids (inci2, incx4, inchi2, etc.) (Yi et al., 2017). Unfortunately, some research found a new resistance gene transfer by plasmids, mcr-2 loci on IncX4 plasmid, which showed 76.7% nucleotide identity to mcr-1 (Xavier et al., 2016). Second, the mutation or deletion of antibiotic binding sites of foodborne pathogens reduces the sensitivity of targeted antibiotics. The major targets of antibiotics in most foodborne pathogens are (i) the cell envelope, (ii) the ribosome, and (iii) nucleic acids (Foster, 2017). Aminoglycosides, tetracyclines, macrolides, and other drugs mainly bind to bacterial ribosomes to inhibit the synthesis of bacterial proteins, resulting in the resistance of bacteria to antibacterial drugs. In general, gyrA, gyrB, parC, and parE constitute the fluoroquinolone antibiotic resistance determining region, and mutation of gyrA leads to a change in the antibiotic binding site, resulting in the development of resistance to antibiotics (Wiuff et al., 2000). Under antibiotic pressure, surviving Salmonella produced the nucleotide base mutation, carrying genes of other Salmonella, which may be a homology relationship, and the resistance genes could escape from CRISPR structural immunity (Piddock, 2002). The DNA of Salmonella possessed single nucleotide polymorphism, which was able to produce resistance to any antibiotic. For example, the polymorphism of the TLR4 gene affected the recognition of Salmonella lipopolysaccharide by toll-like receptor 4, leading to positive or negative effects on human or animal resistance to Salmonella infection (Li et al., 2017b). However, it was difficult to determine whether the pathogenic bacteria were resistant at the beginning, and there would be some mutation sites that were not obvious. It was impossible to distinguish whether the drug resistance was caused by site Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 mutations or other reasons in clinical situations. A previous study reported the directed evolution with random genomic mutations, which could predefine the possibility of mutations in the whole genome sequence of pathogenic bacteria with a million-fold mutation rate, provide a rapid identification of bacterial drug resistance research with the help of this research, and distinguish single drug-resistant bacteria from multidrug-resistant bacteria (Nyerges et al., 2018). The mutant drug resistance gene might cover a wide range of different pathogenic bacteria along with the transmission of the plasmid. Third, the active efflux pump of Salmonella is mainly AcrAB-TolC of the resistance- nodulation-division family. Overexpression of efflux pump regulated genes would lead to drug resistance, recognizes multiple substrates and is associated with multidrug resistance (MDR) (Blair et al., 2014). AcrAB-TolC is composed of periplasmic protein AcrA, efflux transporter AcrB and outer membrane channel protein TolC, which are able to discharge antibiotics outside the cell through the central channel (Tikhonova and Zgurskaya, 2004). After the induction of a Salmonella ciprofloxacin highly resistant strain (resistance value 256 μg/mL) through inactivation of AcrB, the resistance value dropped to 0.25 μg/mL (Siew et al., 2007). What is more, RamA is an AraC/XylS transcription activator and homologue of MarA and regulates the expression of genes encoding the AcrAB-TolC MDR efflux pump (Abouzeed et al., 2008). Similar to E. coli and other Enterobacteriaceae (Figure 3), ramA also regulates the expression of acrAB and tolC (Ricci and Piddock, 2009). In addition, point mutations in ramR, which is the repressor of ramA transcription, as well as ramA and acrB overexpression have all been associated with multidrug resistance (Abouzeed et al., 2008; Ricci and Piddock, 2009). Another study has confirmed that the separation of MarR from the promoter region may promote the expression of MarA, and the MarA protein could activate Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 acrAB and micF and trigger Salmonella typhimurium MDR (Alekshun and Levy, 1997; Prouty and A., 2004). Telke et al. (2017) found that the expression of soxRS may activate the upregulation of acrAB, contributing to the resistance to colistin. In addition, there were distinctions in efflux pump regulatory genes among Bacteroidetes, such as Klebsiella pneumoniae, romA, and rarA, which could regulate acrAB (Rosenblum et al., 2011). Therefore, as an important resistance mechanism, the acrAB pump needs further investigation to solve the problem of antibiotic resistance in Salmonella. A platform based on efflux pump gene expression may be a promising rapid detection method for foodborne pathogens. Finally, there were changes in the bio-membrane permeability. Generally, drugs entering pathogenic bacteria require passage through outer membrane porin (OMP) on the cell membrane (Masi et al., 2019). However, the abnormality in OMP would cause the permeability of bacterial cell membrane to affect the drug resistance of pathogenic bacteria (Hu et al., 2011). Compared to the resistant and sensitive strains of ciprofloxacin, the expression of outer membrane porins F (OmpF) in the resistant strains decreased significantly (Du et al., 2020). Interestingly, OmpF effectively controlled the entry and exit of antibiotics and only allowed antibiotics to enter slowly, which improved the drug resistance rate (Delcour 2009). The loss of OmpC protein led to carbapenem resistance, while OmpC , c d OmpF , and Omp proteins determined the resistance mechanism of Salmonella membrane porin (Vergalli et al., 2020). However, OMP did not play a separate role in the mechanism of drug resistance. Indeed, it regulated the expression levels of ompD and acrD and controlled the penetration of antibiotics to produced drug resistance (Hu et al., 2009). Particularly, OmpF, a Salmonella drug channel, was regulated by MarA and SoxS proteins. Specifically, Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 the expression of marA or soxS activated the transcription of MicF (antisense RNA of OmpF RNA) and prevented the translation of OmpF mRNA, which improved the drug resistance rate by the degradation of OmpF RNA (Hartog et al., 2008). A previous research demonstrated that an OMP-based typing method quickly detected the drug resistance of Salmonella and the expression of OMP protein (Kumar et al., 2019). In general, the above four modes constituted the antibiotic resistance mechanism of Salmonella. The increase in antibiotic dose changed the antibiotic resistance of Salmonella, resulting in resistance gene mutation, which limited the effect of antibiotics. What is more, molecular biology is also committed to exploring better mitigation measures. Therefore, the investigation of antibiotic resistance genes and CRISPR-Cas mechanism may solve these problems. 3. Salmonella resistance to different antibiotics 3.1. Resistance to quinolones and fluoroquinolones Salmonella showed resistance to fluoroquinolones. For example, the fluoroquinolones resistance determining region was composed of gyrA, gyrB, parC, and parE, which played a role in antibiotic resistance by inhibiting the gyrase of Gram-negative bacteria and topoisomerase IV of Gram-positive bacteria (Akiba et al., 2007). Another study demonstrated that the ser83 subunit of Salmonella gyrA site was mutated to phenylalanine and then showed resistance to naphthyridic acid (MIC 256 µg/mL) and ciprofloxacin (MIC 0.25-2 µg/mL); similarly, the asp87 site was mutated into glycine and tyrosine (MIC 256- 512µg/mL of naphthyridic acid and MIC 0.12-0.5 µg/mL of ciprofloxacin) (Chen et al., 2007; Fàbrega et al., 2008). Some researchers suggested that the resistance of Salmonella to naphthyridic acid, enrofloxacin, ciprofloxacin, ofloxacin and levofloxacin and the proportion Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 of antibiotic-resistant strains were 90.2%, 39.0%, 41.5%, 29.3%, and 26.8%, respectively (Fàbrega et al., 2008). Antibiotic resistance in Salmonella should not be underestimated. In addition, researchers have identified a new gene regulatory region of Salmonella resistance to antibiotics called plasmid mediated quinolone resistance (PMQR) (Martínez- Martínez et al., 1998). Moreover, a multi-antibiotic resistant plasmid from clinical isolates of Klebsiella pneumoniae was able to increase Salmonella resistance to fluoroquinolones. The multi-antibiotic plasmid consisted of qnrA, qnrB, qnrC, qnrD and qnrS (Nordmann, 2006), and each family contains different subunits and mutants, such as QnrB1, QnrB7, and QnrB17 (Jacoby et al., 2008). However, Salmonella and Escherichia coli isolated from human feces carried plasmids with qnrA, qnrB, and qnrS resistance genes, which showed resistance to fluoroquinolones (Doma et al., 2020). Apart from PMQR, another resistance mechanism of quinolones and fluoroquinolones was the production of a modified aminoglycoside acetyltransferase (AAC(6)-b-cr), which reduced the activity of the compound through enzyme modification and the activity of ciprofloxacin by n-acetylation of the amino nitrogen on the piperazinyl substituent (Robicsek et al., 2006). Salmonella plays an essential role in antibiotic resistance through AcrAB-TolC efflux system. Depending on the expression of acrAB, the damage of eight transporters would result in the construction of multigene deletion mutants (ΔacrA, ΔacrB, ΔtolC, ΔacrAB, ΔacrEF, ΔacrD, ΔmdsABC, ΔmdtABC, ΔemrAB, ΔmacAB, ΔmdfA, ΔmdtK, ΔacrAB-ramA, ΔacrAB-marA, and ΔacrAB-soxS), and ramA and marA contributed to coordinating the regulation feedback mechanism of AcrAB-Tolc and 8 other functional efflux pumps (Zhang et al., 2018). Additionally, Salmonella isolated from the environment generally gained PMQR genes (59%), including qnrS1, aac(6)-1b-cr, qnrB19, and qnrD genes (Veldman et al., 2011). Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 In addition to the gyrA mutation in the fluoroquinolone determining region (Yang et al., 2012), a common drug resistance gene oqxAB based on chloramphenicol and fluoroquinolone antibiotics was also found in Hong Kong, China (Wong and Chen, 2013). 3.2. Resistance to sulfonamides and trimethoprim Sulfamethoxazole (SMZ) is a synthetic antibacterial drug that has been used clinically for nearly 50 years. The combined application of trimethoprim (TMP) and SMZ enhanced the antibacterial effect and expanded the treatment range (Falagas et al., 2015; Vila-Costa et al., 2017). However, after long-term use, broad-spectrum antibiotics seemed to become less effective. SMZ is usually associated with mutations in the gene encoding dihydrofolate synthase (DHPS). However, clinical antibiotic-resistant mutants possessed additional compensatory mutations in DHPS so that the antibiotic-resistant system worked normally (Sköld 2000). The resistance of Salmonella to sulfonamides drugs was related to the presence of the sul (sul1, sul2 and sul3) genes, which was the encoding forms of DHPS that were not inhibited by antibiotics (Antunes et al., 2005). Moreover, the mechanism of Salmonella resistance to sulfonamides was mainly plasmid transduction. For example, sul1 was lied in 3’-conserved segment (3’-CS) of class 1 integrons (Sköld 2000). However, sul2 exists on antibiotic- resistant plasmids, and Salmonella might exhibit resistance by controlling the expression of plasmids (Enne et al., 2004). The drug resistance gene sul3 came from a wide range of sources and has been detected in large plasmids of different Salmonella (Guerra et al. 2004). Similar to SMZ, trimethoprim also competed with essential folate pathway substrates in bacteria and inhibited dihydrofolate reductase and dihydrofolate reductase variants, which reduced the sensitivity of Salmonella to trimethoprim (Korsak & Krawczyk-Balska 2017). Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Researchers reported mutants of dhfr Ⅰ and dhfr Ⅱ in Gram-negative intestinal bacteria, which made the MIC of TMP 11000 times higher than that of normal (Huovinen 2001). At present, researchers often detect the Sul gene family of Salmonella in meat products (Xu et al., 2020). In research, 58.1% of Salmonella were resistant to SMZ, and 34% were resistant to TMP (Thai et al., 2012b). Among the meat sold in Canada, 110 strains of Salmonella were identified, 71% of which were resistant to SMZ, and sul1, sul2, and sul3 resistance genes were detected in the isolated Salmonella (Aslam et al., 2012). 3.3. Resistance to β-lactams β-Lactams are widely used in foodborne pathogens and have high antibacterial activity. The invention of lactam drugs alleviated the negative reaction of human beings to microbial infection and the embarrassing situation of dealing with pathogens without available antibiotics. The abuse of antibiotics brought about the emergence of extended spectrum β- lactamases (ESBLs) resistant strains. ESBLs hydrolyzed penicillin and cephalosporins (excluding cephalosporins). However, this drug resistance mechanism is not effective for a long time. The hydrolysis efficiency of ESBLs was inversely proportional to that of β-lactam inhibitors, currently known related drug resistance genes include , , , and (Winokur et al., 2000). Resistance genes such as bla (detection rate CTX-M 94.1%), (45%), and (24.1%) were detected in isolates (Kuang et al., 2018). In addition, the monitoring of pAmpC-harboring and ESBL-producing Salmonella has also become particularly important. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 3.4. Resistance to chloramphenicol Chloramphenicol acetyltransferases (CATs) regulated the antibiotic resistance of chloramphenicol resistance in foodborne pathogenic bacteria. Affected by HGT, the cat gene is widely expressed in foodborne pathogenic bacteria. The cat gene of Salmonella was expressed from plasmids of other pathogenic bacteria or mutation, and the cat gene family also contains the cata1, cata2, and variant catB genes (Villa et al., 2002; Chen et al., 2004; Ahmed et al., 2005). The production of new mutations in Salmonella also improved the sensitivity to chloramphenicol. In addition, cmlA and floR effectively inhibited the expression of efflux pump genes, prevented the outward penetration of chloramphenicol and reduced the sensitivity of chloramphenicol (Thai et al., 2012a). Salmonella isolated from meat products contained at least one antibiotic resistance gene, of which 57.1% Salmonella contained floR, 50% contained cmlA1, and 14.3% contained dual antibiotic resistance genes (Thai et al., 2012a). A food sampling inspection in Germany showed that the detection rate of floR gene in antibiotic-resistant Salmonella was the highest (90.9%), while the detection rates of catA and cmlA were lower (Miko et al., 2005). Similarly, the detection frequency of cat1 and cat2 in antibiotic-resistant Salmonella was very low, which explained that they were not the main chloramphenicol resistance regulatory genes (Thong and Modarressi, 2011). As described, antibiotic resistance gene mediation was the main reason why Salmonella was sensitive to chloramphenicol. The above research proved that floR gene may be transferred in Salmonella through HGT, and the HGT frequency of cat1 and cat2 genes was not very high. At present, the main factors of Salmonella resistance come from HGT of plasmids. In addition, the interaction between drug resistance genes and CRISPR immune mechanism needs to be further studied. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 3.5. Brief summary of resistance The mechanism of drug resistance was generally described, and the resistance genes were summarized, which laid a foundation for the follow-up related resistance research. Notably, some other broad-spectrum antibiotic resistance gene loci were statistically analysed (Table 1) (Karczmarczyk et al., 2010; Deekshit et al., 2012). From Table 1, gyrA, gyrB, parC, and parE are the determinants of drug resistance to fluoroquinolones. To date, five different qnr families, each with a different number of alleles (qnrA1-7, qnrS1-4, qnrB1-31, qnrC and qnrD) and oqxAB, have also been widely studied (Table 1). For example, the qnr family is common in poultry, pigs, sheep and turkeys, especially qnrS1 in E. coli isolated from humans, and 59% of Salmonella contains the qnr family (Veldman et al., 2011). Research on the resistance genes of Salmonella to sulfonamides showed that 152 strains of sul1 gene (76%), 74 strains of sul2 gene (37%) and 14 strains of sul3 gene (7%) were detected in 200 resistant strains, and sul1 and sul2 emerged in 24 isolates at the same time (Antunes et al., 2005). However, 34% of foodborne Salmonella were highly resistant to trimethoprim (Thai et al., 2012a). The trimethoprim resistance genes of Salmonella are shown in Table 1, and Deekshit found that Salmonella Weltevreden contained a class 1 integron with a single gene dfrA7 in the integron cassette and Salmonella Newport with dhfrA1(Deekshit et al., 2012). In fact, the study found that there are many kinds of β-lactam antibiotic resistance genes of Salmonella (Table 1). The majority of cephalosporin resistant Salmonella express a broad-spectrum β-lactamase, which can hydrolyze oximine cephalosporins but not cephalosporins (Winokur et al., 2000). In addition, bla , bla , bla and bla β-lactamase genes were validated by PCR, and CMY ctx-m TEM OXA Salmonella containing plasmid-mediated bla gene increased from 0% (0/27) in 2000 and CMY-2 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 2001 to 75% in 2004 and 2005, which especially had a higher frequency of infection than human with non-bla (Zaidi et al., 2007). The determinants of chloramphenicol CMY-2 resistance in Salmonella include catA1, floR and cmlA1. Although compared with the three antibiotics, the resistance rate of chloramphenicol is limited, and the induction effect on Salmonella resistance and antibiotic membrane activity is weak (Lorenzo 2019). 4. Study on CRISPR-Cas and antibiotic resistance of Salmonella Typically, cas genes are used to express DNA cleavage enzymes, combined with PAM localization, and spacers are used as templates to exert immune effects. Strains without CRISPR-Cas are more prone to HGT of antibiotic-resistant plasmids, which might be related to the immune mechanism of CRISPR-Cas (Palmer and Gilmore 2010). For example, Enterococcus faecalis obtained antibiotic resistance genes (AGRs) by escaping CRISPR-Cas immunity. For this reason, strains without CRISPR loci were more likely to obtain antibiotic resistance genes than strains with complete CRISPR loci. Salmonella contains complete CRISPR-Cas, and two antibiotic resistance genes, bla CMY-2 and aac(6’)-Iaa, were obtained from the incll plasmid (Monte et al., 2021). In addition, phages slowly helped Salmonella cope with the threat of antibiotics due to the transduction of antibiotic-resistant plasmids. The reason for the decreased sensitivity of Salmonella to ciprofloxacin was that phage DT104 carried ARGs. The abovementioned phenomenon was explained by Salmonella apparently receiving ARGs and appearing in CRISPR-Cas as a new spacer, resulting in the emergence of antibiotic resistance (Threlfall et al., 2000). CRISPR-Cas typing was positively correlated with antibiotic resistance corresponding to serotypes in serotype verification (Hoelzer et al., 2010). At present, experiments show that the pressure of wild strains on antibiotics decreased and the sensitivity of antibiotics Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 increased. The clinical isolated Salmonella showed that the CRISPR-Cas structure had significant resistance to broad-spectrum antibiotics (DiMarzio et al., 2013). CRISPR-Cas usually coexists with antibiotic resistance genes in Salmonella. CRISPR-Cas not only obtained antibiotic resistance genes as new spacers but also adopted other genes beneficial to itself (Shehreen et al., 2019). One study showed that self-targeting spacers accounted for 0.4% of all spacers (including the vast majority of spacers without assignable original spacers) and appeared in 18% of the contained prokaryotic genome (Stern et al., 2010). The above study showed that the spacers abandoned the old sequences and adopted the new sequences, and there was the possibility of adding dissociative drug resistance genes to spacers. Studies have shown that Cas9 protein had enzymatic digestion of free exogenous RNA, even without PAM (O'Connell et al., 2014; Rousseau et al., 2018). Therefore, CRISPR-Cas of Salmonella also acted on the RNA products of resistance gene expression. In particular, Streptococcus thermophilus CRISPR1-Cas also obtained naturally antibiotic resistance genes of plasmids (Garneau et al., 2010). In addition, the spacers of Klebsiella pneumoniae found to be β- lactams, quinolones, and aminoglycosides were partially resistant fragments (Wang et al., 2020). With the emergence of resistance gene fragments in the spacers, perhaps the older spacers were removed, but the drug resistance of bacteria was not affected and was still in the drug resistance stage. Under the pressure of antibiotic selection, the CRISPR-Cas of Salmonella may be mutated, allowing the bacteria to obtain foreign resistance genes and survive (Li et al., 2021). The spacers mutation made the free resistant fragments unaffected by CRISPR-Cas. For example, E. coli retained the same spacers as the foreign plasmids, indicating that they had ancient contact with the plasmids, although spacers expression was no longer active (Touchon et al., 2012). The resistance gene fragments in the spacers would mutate autonomously under environmental pressure and weaken the intervention of Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 CRISPR-Cas on HGT. CRISPR-Cas in mammalian intestinal flora prevented the transmission of antibiotic-resistant plasmids, such as Enterococcus faecalis (Price et al., 2019a). On the other hand, type I-E of Klebsiella pneumoniae might interfere with the acquisition of plasmids containing antibiotic resistance genes to keep the strain sensitive to antibiotics (Li et al., 2018). Compared with CRISPR-Cas of Klebsiella pneumoniae, Salmonella was also type Ⅰ -E with the same antibiotic sensitivity mechanism. No plasmids or phage-targeted spacers were identified to match any drug resistance genes, so most of the drug resistance gene fragments in the spacers came from free fragments. Although anti-CRISPRs had evolved to provide CRISPR-Cas advantages to virulent and temperate phages, they evolved in a variety of ways (Landsberger et al., 2018). Anti-CRISPR benefited ARGs while reducing the defense ability of CRISPR-Cas against phages. Under antibiotic selection, Salmonella inserted the spacers into CRISPR-Cas to complete the immune response. Therefore, ARGs were obtained only in cells without mobile genetic elements targeting spacers, which was the reason why CRISPR-Cas targeted exogenous DNA, not ARGs (Shehreen et al., 2019). CRISPR-Cas and ARGs of bacteria could not exist at the same time because ARGs were incorporated into the spacers of CRISPR-Cas. In summary, ARGs might also be recognized and degraded by Cas protein due to bacterial immunity. Given the above, based on the theory that ARGs and CRISPR-Cas cannot co-exist, research introduced the use of CRISPR-Cas to solve the problem of bacterial antibiotic resistance (Aslam et al., 2020). Moreover, CRISPR was more commonly used to analyse Salmonella serotypes with advantages compared with pulsed field gel electrophoresis and multi-locus sequence analysis (DiMarzio et al., 2013; Vosik et al., 2018). Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 CRISPR-Cas9 gene editing technology was the most important discovery in this century. It showed that mature crRNA formed a special double-stranded RNA structure with tracr- RNA through base complementary pairing, which guided Cas9 protein to cause double- strand breaks in the target DNA (Gupta et al. 2019). CRISPR-Cas9 removed the plasmids carrying mcr-1 in intestinal pathogens and improved the sensitivity of intestinal bacteria to colistin (Dong et al., 2019). After inserting artificially edited Cas9, which acted on the virulence genes of Staphylococcus aureus, and destroyed the plasmids carrying antibiotic resistance genes (Bikard et al., 2014). Especially, CRISPR-Cas9 antimicrobials function in vivo to kill S. aureus in a mouse skin colonization model. In addition, the artificially edited CRISPR-Cas9 purposefully acted on the elimination of antibiotic-resistant plasmids and phage invasion while protecting antibiotic-sensitive bacteria (Yosef et al., 2015). This strategy could be applied to the fermentation of hospital hand sanitizer and engineered bacteria. Combined with previous research, it is speculated that the coupling mechanism is the best way to provide CRISPR-Cas9 antibacterial agent to bacteria (Dong et al., 2019). Based on the above theory, the inhibition of the HGT effect by artificial CRISPR-Cas9 transduction increased the sensitivity of Salmonella to antibiotics. The crRNA and tracr-RNA synthesize polymer small guide RNA (sgRNA), which was designed to eliminate antibiotic resistance genes from bacterial DNA (Gomaa et al., 2014). Moreover, CRISPR-Cas9 reduced the occurrence of antibiotic resistance in a sequence-specific manner (Rodrigues et al., 2019). For instance, Campylobacter lacks cas9, which improves its sensitivity to antibiotics and the level of HGT (Shabbir et al., 2018). Cas9 deletion increased the antibiotic sensitivity of C. jejuni, which may be related to cas9 preventing the HGT of resistance genes. Given the above, plasmids or phages carryingCRISPR-Cas9 system with specific sequences can selectively remove drug-resistant bacteria from complex strains (Bikard et al., 2014). As Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 described, based on drugresistant Salmonella isolated from clinical or poultry feed, it is possible to eliminate drug-resistant genes by CRISPR-Cas9 plasmids, and even the plasmids contain multiple resistance gene templates. If the restriction of PAM codon is overcome, it will also be possible to eliminate multiple resistance genes at one time. Moreover, similar to the construction of E. coli movable CRISPR-Cas genetic elements (Yosef et al., 2015), cas gene clusters were combined with lysogenic bacteriophages. The cse1, cse2, cas7, cas5, cas6 and cas3 contained in Salmonella CRISPR-Cas were enough to knock out the target resistance genes. A series of pIS26-CRISPR/Cas9 suicide plasmids were constructed, and specific guiding RNAs targeting Incx4, Inci2 and Inchi2 plasmid replication genes and drug resistance genes mcr-1, blakpc-2 and blandm-5 were designed, and the plasmid of CRISPR- Cas9 based on IS26 can be effectively used as an immune system to prevent the acquisition of exogenous resistant plasmid again (He et al., 2021). Therefore, it is feasible to mutate the drug resistance gene fragments of CRISPR-Cas9 plasmids or phages, and it has a better treatment effect on the drug resistance of pathogenic bacteria. At present, the application of plasmid or phage containing CRISPR-Cas9 in Salmonella is limited, and a lot of studies need to be carried out. In particular, Price et al. (2019b) found that CRISPR-Cas effectively prevented the HGT effect of antibiotic resistance genes in mammalian intestinal flora. With the continuous exploration of CRISPR-Cas, more strategies may emerge to inhibit food safety problems caused by foodborne pathogens. In addition, CRISPR-Cas is not only directly related to drug resistance but also slightly related to quorum sensing (QS) of pathogenic bacteria. For example, there has also been some progress in researches on the relationship between QS and CRISPR. For instance, research showed that when the cas3 gene was missing in the CRISPR structure of Salmonella, the lsrFGBE genes related to QS were upregulated, and the biofilm forming ability decreased (Cui et al., 2020). Moreover, the Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 absence of AI-2 affects the immune acquisition of spacers in Salmonella CRISPR structure. QS pro-interfering enzyme in Pseudomonas aeruginosa significantly changed the expression of CRISPR-Cas genes (Mion et al., 2019). 5. Conclusions The food safety problem caused by Salmonella is becoming increasingly serious. The independent CRISPR-Cas structures among strains were significantly different in response to the HGT of pathogenic bacteria. HGT is the main transmission mechanism of drug resistance genes. The analysis showed that it was easier to obtain the resistant fragments without CRISPR locus, such as Enterococcus faecalis. It can be concluded that resistant genes in spacers mainly come from the free fragments and phages in the environment. Therefore, the spacers in CRISPR-Cas were different between drug-resistant and nondrug-resistant strains. In general, there was a close relationship between CRISPR-Cas and drug resistance in Salmonella, which was speculated to interfere with the expression of drug-resistant plasmids and cytoplasmic free resistant fragments. In addition, gene editing based on CRISPR-Cas9 accurately mutates the resistance genes in drug-resistant Salmonella. The review lays the foundation of food safety at the molecular level and provides a new research idea to solve the antibiotic resistance of foodborne pathogens. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Conflict of interest There are no conflicts to declare. Funding This study was supported by the Graduate Innovative Fund of Wuhan Institute of Technology (No. CX2020347) and the Key projects of science and technology research plan of Hubei Provincial Department of Education (D202015001), China. Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 References Abouzeed, Y. M., Baucheron, S., Cloeckaert, A. (2008). ramR mutations involved in efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium. Antimicrobial agents and chemotherapy, 52(7): 2428-2434. Ahmed, A. 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Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Table 1 Examples of drug-resistant Salmonella isolated from food Type of antibiotic Resistance gene Reference Quinolones gyrA, gyrB, parC, parE Veldman K.et al and Karczmarczyk and qnrB, qnrD, qnrS, oqxAB M.et al Fluoroquinolones Sulfonamides sul1, sul2, sul3 Antunes P.et al and Guerra B.et al dfrA1, dfrA12, dfrV, dhfr1, dhfrV, Thai TH Chen S and Deekshit VK.et Trimethoprim dhfrA7, dhfr12, dhfr13, dhfr17 al , , , β-lactams , , , Aslam M and Chen S.et al Chloramphenicol catA1, floR, cmlA1 Miko A and Deekshit VK.et al Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Accepted Manuscript Downloaded from https://academic.oup.com/fqs/advance-article/doi/10.1093/fqsafe/fyac035/6585011 by DeepDyve user on 17 May 2022 Accepted Manuscript

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Published: May 12, 2022

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