Characterization of the Role of Two-Component Systems in Antibiotic Resistance Formation in Salmonella enterica Serovar Enteritidis

ABSTRACT The two-component system (TCS) is one of the primary pathways by which bacteria adapt to environmental stresses such as antibiotics. This study aimed to systematically explore the role of TCSs in the development of multidrug resistance (MDR) in Salmonella enterica serovar Enteritidis. Twenty-six in-frame deletion mutants of TCSs were generated from S. Enteritidis SJTUF12367 (the wild type [WT]). Antimicrobial susceptibility tests with these mutants revealed that 10 TCSs were involved in the development of antibiotic resistance in S. Enteritidis. In these 10 pairs of TCSs, functional defects in CpxAR, PhoPQ, and GlnGL in various S. Enteritidis isolates led to a frequent decrease in MIC values against at least three classes of clinically important antibiotics, including cephalosporins and quinolones, which indicated the importance of these TCSs to the formation of MDR. Interaction network analysis via STRING revealed that the genes cpxA, cpxR, phoP, and phoQ played important roles in the direct interaction with global regulatory genes and the relevant genes of efflux pumps and outer membrane porins. Quantitative reverse transcription-PCR analysis further demonstrated that the increased susceptibility to cephalosporins and quinolones in ΔphoP and ΔcpxR mutant cells was accompanied by increased expression of membrane porin genes (ompC, ompD, and ompF) and reduced expression of efflux pump genes (acrA, macB, and mdtK), as well as an adverse transcription of the global regulatory genes (ramA and crp). These results indicated that CpxAR and PhoPQ played an important role in the development of MDR in S. Enteritidis through regulation of cell membrane permeability and efflux pump activity. IMPORTANCE S. Enteritidis is a predominant Salmonella serotype that causes human salmonellosis and frequently exhibits high-level resistance to commonly used antibiotics, including cephalosporins and quinolones. Although TCSs are known as regulators for bacterial adaptation to stressful conditions, which modulates β-lactam resistance in Vibrio parahaemolyticus and colistin resistance in Salmonella enterica serovar Typhimurium, there is little knowledge of their functional mechanisms underlying the development of antibiotic resistance in S. Enteritidis. Here, we systematically identified the TCS elements in S. Enteritidis SJTUF12367, revealed that the three TCSs CpxAR, PhoPQ, and GlnGL were crucial for the MDR formation in S. Enteritidis, and preliminarily illustrated the regulatory functions of CpxAR and PhoPQ for antimicrobial resistance genes. Our work provides the basis to understand the important TCSs that regulate formation of antibiotic resistance in S. Enteritidis.

and nonpathogenic bacteria, but their regulons vary in different bacteria, such as S. Typhimurium, E. coli, P. aeruginosa, Yersinia pestis, and Mycobacterium tuberculosis (20)(21)(22)(23)(24). Previous comparative genomic studies have suggested that regulons exhibit considerable plasticity across the evolution of bacterial species (25,26). In this regard, it has more practical significance to systematically study the contribution of TCSs, including CpxAR and PhoPQ, in MDR S. Enteritidis.
To date, most isolates of MDR S. Enteritidis from both food and clinical settings exhibit high resistance to clinically recommended antibiotics (e.g., quinolones and cephalosporins), thus limiting the clinic treatment options. Anti-signal transduction is an alternative therapy to defeat antibiotic-resistant bacteria, and thus TCSs are considered attractive antibacterial targets (27). Therefore, the systematic identification and elucidation of TCSs that contribute to MDR are crucial for the prevention and control of antibiotic resistance in S. Enteritidis. We have recently reported the complete genome of a sequence type 11 (ST11) S. Enteritidis isolate, SJTUF12367, with an extensive antibiotic resistance profile and abundant ARGs (28). S. Enteritidis ST11 strains have been reported to be a globally dominant clone, in which MDR phenotypes are common (29). The purpose of the present work was to systematically characterize the role of the TCSs in development of MDR in S. Enteritidis SJTUF12367 and other related clinical isolates through gene knockout and antimicrobial susceptibility tests and to explore the regulatory mechanisms of two important TCSs (CpxAR and PhoPQ) against quinolones and cephalosporins through interaction network analysis, quantitative reverse transcription-PCR (qRT-PCR), and membrane perturbation analysis. This work will contribute to a better understanding of antibiotic resistance mechanisms as well as a basis for combating antibiotic resistance in S. Enteritidis.

RESULTS
Functional defects in 10 pairs of TCSs resulted in an increase in S. Enteritidis susceptibility to antibiotics. Twenty-six RR genes were replaced by a flippase recognition target (FRT) site-flanked hph cassette that contains a hygromycin resistance gene as selection marker in S. Enteritidis SJTUF12367, resulting in 26 TCS deletion mutants (see Fig. S1 in the supplemental material). The growth curve of these mutants was measured in LB. As shown in Fig. 1A, compared to the wild type (WT), the DglnG::hph mutant exhibited delayed growth (P , 0.05) during the logarithmic phase and the DphoP::hph mutant displayed a little lower culture density during the stationary phase, but there was no apparent change in bacterial growth rate in the rest of the TCS mutants. It was also found that there was no significant difference in the growth rates between the DglnG and WT strains when 0.4% glutamine was added to the culture: i.e., the glutamine could recover the delayed growth in the DglnG strain, indicating the importance of GlnGL in regulation of nitrogen source synthesis (Fig. 1B).
The MICs of the WT and 26 mutants against eight classes of antibiotics were determined, which covered the most common clinical and veterinary antibiotics. The results in Table 1 show that MIC values of 10 TCS mutants were significantly altered, while those of the other 16 TCS mutants were not apparently altered (data not shown). The DarcA::hph mutant showed 2-and 4-fold decreases in the MICs of tetracycline (TET) and azithromycin (AZI), whereas there were 2-and 4-fold increases in the MICs of streptomycin (STR) and gentamicin (GEN), respectively, compared to the WT. In addition, 2and 16-fold decreases in the MICs of cefotaxime (CTX) and colistin (COL), respectively, for the DbasR::hph mutant (basR is also known as pmrB), a 4-fold decrease in the MIC of GEN for the DbaeR::hph mutant, a 4-fold increase in the MIC of GEN for the DnarP::hph mutant, a 2-fold decrease in the MIC of CTX for the DompR::hph mutant, a 2-fold decrease in the MIC of ceftriaxone (CRO) for the DyehT::hph mutant, and a 2-fold decrease in the MICs of ciprofloxacin (CIP) and ofloxacin (OFX) for the DuvrY::hph mutant were also observed.
It was found that deletion of the gene cpxR, phoP, or glnG influenced the susceptibility of S. Enteritidis to at least three classes of antibiotics. Compared to the WT, there was a 16-fold decrease in the MIC of colistin in the DphoP::hph mutant and there was a 2-to 4-fold decrease in the MIC of aminoglycosides (STR, GEN, and kanamycin [KAN]) in the DcpxR::hph mutant. Both DphoP::hph and DcpxR::hph strains showed 2-to 8-fold decreases in the MICs of cephalosporins (CAZ, CRO, CTX, and cefepime [FEP]) and quinolones (NAL, CIP, and OFX). Likewise, there was a 2-to 8-fold reduction in resistance to antibiotics, including piperacillin (PIP), CAZ, CRO, CTX, FEP, STR, GEN, AZI, TET, and sulfamethoxazole-trimethoprim (SXT) in the DglnG::hph mutant. These results were also consistent with the resistant phenotype in the hph cassette-free DcpxR, DglnG, and  (Table S5) targeting corresponding positions of the sequence (not to scale). Amplicon sizes are shown in between for each pair of primers. (b) Mutant confirmation by PCR. The primers used to check phoP, phoP plus flanking sequences, and the hph cassette are shown in panel a. Primers EBGNHe-5/EBGh3-3 and PR1655/PR1656 were used for determination of pKOBEG-Apra and pFLP2-Apra, respectively. Expected amplicon sizes are given in parentheses.     (Table 1). CpxAR, PhoPQ, and GlnGL played an important role in developing MDR in different S. Enteritidis isolates. To determine the roles of CpxAR, PhoPQ, and GlnGL in regulation of drug resistance of various S. Enteritidis isolates, six clinical MDR isolates (SE1 to -6), which exhibited resistance to cephalosporins and nalidixic acid and/or ACSSuT antimicrobial resistance profiles (Table S1), were used to construct deletion mutants with single gene deletions of cpxR, phoP, and glnG, respectively. The resulting deletion mutants were verified by PCR and sequencing (Fig. S3). The MICs for SE1 to -6 and their mutants against quinolones, cephalosporins, aminoglycosides, colistin, and tetracycline were then determined. As shown in Table 2, these wild-type (WT) isolates exhibited resistance to CAZ, CRO, CTX, FEP, and NAL. Among the 6 cpxR deletion mutants, 4 showed 2-to 8-fold decreases in the MICs of CAZ, CRO, OFX, NAL, STR, KAN, and GEN compared with their parental strains. In addition, all 6 of the phoP deletion mutants showed 2-to 16-fold decreases in the MICs of CAZ, CRO, FEP, CIP, OFX, NAL, and COL. Most of the DglnG::hph mutants showed 2-to 8-fold decreases in the MICs of CAZ, CRO, FEP, CIP, and TET, and a few mutants exhibited higher susceptibility to OFX, NAL, STR, or KAN than their parental strains. The changes in antibiotic sensitivity of these mutants were consistent with those caused by cpxR, phoP, or glnG deletion in S. Enteritidis SJTUF12367. Altogether, CpxAR, PhoPQ, and GlnGL all modulated resistance of S. Enteritidis to quinolones and cephalosporins, in which CpxAR also facilitated resistance to aminoglycosides, PhoPQ conferred resistance to colistin, and GlnGL mediated resistance to aminoglycosides and tetracycline. These results demonstrated that CpxAR, PhoPQ, and GlnGL contributed to the development of multiple-drug resistance in S. Enteritidis MDR isolates. CpxAR and PhoPQ directly interacted with ARGs. To characterize the interactions between important TCSs (cpxAR, phoPQ, and glnGL) and ARGs, the ARGs of the SJTUF12367 genome were collected from the 3 databases PATRIC, CARD, and NDARO and analyzed. A total of 127 ARGs (33 in PATRIC, 39 in CARD, and 55 in NDARO) were obtained, and 78 nonredundant ARGs were screened out. Finally, 55 ARGs (including cpxAR and phoPQ) showed interactions in the STRING database along with 163 functional interactions, and three densely interconnected clusters (designated C1 to -3) were obtained via Cytoscape ( Fig. 2A). However, string analysis showed that glnGL had no interaction links with ARGs.
These ARGs were responsible for various resistance mechanisms, including multidrug efflux pumps (19 genes), alteration in drug targets (25 genes), enzymatic inactivation/degradation (2 genes), drug permeability restriction (1 gene), and MDR mechanisms (8 genes) (Fig. 2B). Additionally, functional enrichment analysis showed that C3 genes (phoP, phoQ, ARGs of S. Enteritidis. The interaction between two genes (nodes) is represented in colored lines (edges), and it represents evidence derived from various sources (high-throughput experimental data, mining of databases, literature, and analyses of coexpressed genes). Three gene clusters are marked as C1 to C3. Each cluster has been given a different color, and the genes not included in clusters are shown in pale yellow. (B) Classification of drug resistance mechanisms of ARGs. ARGs involved in each mechanism are represented in different colors based on the drug resistance mechanism. (C) Extended network of C3 genes. The interaction network of C3 ARGs was extending to 38 node genes and 264 interaction edges, and the average interaction score of each node gene was 13.9. C3 genes are shown as ellipses, whereas functional partners are shown as diamonds and hexagons according to their gene function. The nodes were given different colors based on their function category.
Role of TCSs in Drug Resistance of S. Enteritidis mSphere crp, marR, acrA, mgrB, emrA, and cpxR) were enriched in the pathway of cationic antimicrobial peptide resistance (eco01503), response to stimulus (GO:0050896), drug transmembrane transporter activity (GO:0015238), and DNA binding (GO:0003677), most of which were involved in the MDR mechanisms (data not shown).
To explore the ARGs' correlation with cpxAR and phoPQ, the extended network with C3 genes was analyzed. The functional partners of C3 genes were classified into class I and II based on their functions via STRING clusters ( Fig. 2C and Table S2). The genes in class I (CL:46) consisted of rpo, rps, and rpl operons, which encoded RNA polymerase (RNAP) and the 30S and 50S ribosomal subunits, respectively, and were responsible for ribosome assembly. Another observation in class II (CL:3574) showed a high number of interconnections among efflux pump genes (acrAB, acrD, macB, and emrAB), global regulator genes (marA and crp), and the marRAB operon repressor gene (marR), which were enriched in the pathway of response to antibiotic (GO:0046677) and transmembrane transporter complex (GO:1902495). In Salmonella, the tripartite complex AcrAB-TolC extruded multiple antibiotics, such as b-lactams, quinolones, and tetracyclines, and gave rise to MDR. Other efflux pumps (EmrAB, AcrD, and MacB) also expelled specific drugs and would in a way compensate for the loss of function of the inactivated AcrAB pump (30). The global transcriptional regulators, not limited to MarA, upregulated acrAB expression and played an important role in compensatory regulation at a global level (30). Therefore, the class II genes were responsible for antibiotic transmembrane export by enhancing efflux pump activity.
The interactions of cpxAR and phoPQ with global regulatory factors were presented in the C3 extended networks, which included the interconnections among cpxAR, marA, tolC, and crp and the interactions between phoPQ and crp. These association cues were somehow coordinated through a coupling of CpxAR or PhoPQ signaling. It has been reported that CpxAR responded to envelope damage, followed by modulation of transcription of marA, activation of acrAB-tolC efflux, and alteration of expression of some outer membrane (OM) porin genes (ompC and ompF) in E. coli (31). The cAMP receptor protein (CRP) encoded by crp could recognize the RNA polymerase (RNAP)-binding site and modulate expression of hundreds of genes through interfacial interactions with RNAP (32); it was found that loss of crp resulted in an increase in resistance to fluoroquinolones in S. Typhimurium via reduced expression of OM porin genes (ompA, ompC, and ompF) and increased expression of an efflux pump gene (acrB) (33). Therefore, the interaction results proposed that CpxAR and PhoPQ of S. Enteritidis might affect the function of global regulators to enhance the fine-tuning of efflux pump and OM porin genes.
Deletion of phoP/cpxR resulted in an increase in cell membrane permeability. The relative mRNA expression of a series of ARGs among WT and DphoP/DcpxR cells was determined via quantitative reverse transcription-PCR (qRT-PCR). In this work, ceftazidime (CAZ) and nalidixic acid (NAL) were used as the representatives of quinolones and cephalosporins, respectively. The subinhibitory concentrations of 2 mg/L CAZ (1/ 16 MIC) and 32 mg/L (1/8 MIC) NAL were selected because all strains could grow at these concentrations, and the population density of DcpxR/DphoP mutants was able to reach a WT level (Fig. S4).
The porins OmpA, OmpC, OmpD, and OmpF were abundant in bacterial outer membrane, among which OmpC and OmpF were linked directly to ciprofloxacin resistance (34), while OmpA was a large porin that allowed for slow diffusion of molecules at a rate 50 times lower than that of OmpF (35). As shown in Fig. 3A, the transcriptional levels of the ompA, ompC, ompD, and ompF genes were measured at the bacterial exponential growth phase (2 h) and the early stationary phase (8 h or 10 h) under the unstressed condition. Compared to WT cells, the transcriptional levels of ompC and ompA genes were not significantly different among all mutants. However, deletion of cpxR or phoP resulted in higher expression levels of ompD and ompF genes than the WT (P , 0.05) after 10 h, which indicated that the homeostasis of bacterial envelope was disturbed.
To test the hypothesis that the DcpxR/DphoP mutants were more permeable to small-molecule antibiotics than the WT, we treated bacterial cells with subinhibitory concentrations of ceftazidime (2 mg/L) or nalidixic acid (32 mg/L). As expected, the differences in the expression of porins between the WT and mutants were enlarged after adding antibiotics (Fig. 3A). The expression levels of the ompC, ompD, and ompF genes were significantly (P , 0.01) higher in DcpxR/DphoP mutants than in the WT at the early stationary phase. The expression level of the ompA gene did not apparently differ Permeabilization activity against membranes of S. Enteritidis wild-type and DcpxR/ DphoP mutants was determined using 1-N-phenylnaphthylamine (NPN) uptake assays (36). As shown in Fig. 3B, the fluorescence intensity of NPN was not significantly changed among WT cells not under stress or with CAZ and NAL added, indicating that subinhibitory concentrations of antibiotics did not destroy the OM permeability of WT. In addition, the fluorescence intensities of WT, DphoP, and DcpxR cells were similar without antibiotic treatment. However, compared to WT cells, the NPN fluorescence intensity was significantly increased in DcpxR and DphoP cells after adding antibiotics and was especially markedly increased in the DcpxR and DphoP cells under the ceftazidime condition (P , 0.01). The difference between NAL-and CAZ-treated cells could arise from severe membrane damage by b-lactam-mediated perturbation of peptidoglycan biosynthesis, which led to destabilization or lysis of the cellular envelope. Overall, CpxAR and PhoPQ could maintain and modify bacterial membrane permeability by regulating expression of OM porins and result in an increase in the resistance of S. Enteritidis to quinolones and cephalosporins.
CpxAR and PhoPQ strengthened the specific efflux pump activity. To characterize the role of PhoPQ and CpxAR in the function of efflux pumps, qRT-PCR was carried out with or without antibiotic to test the transcription characteristics of 4 families of efflux pump genes, including the resistance-nodulation-cell division (RND) family pumps (AcrA, AcrD, and MdsA), the major facilitator superfamily (MFS) pump (EmrA), the multidrug and toxic compound extrusion (MATE) family pump (MdtK), and the ATP-binding cassette (ABC) family pump (MacB). After an incubation for 10 h with nalidixic acid, DphoP mutant cells were shown to have significantly reduced expression levels of the macB and mdtK genes, while the DcpxR mutant had lower expression levels of the acrA and acrD genes (P , 0.001) than the WT (Fig. 4A and B). After ceftazidime treatment for 8 h, there was no significant alteration in the expression levels of macB and mdtK genes in the DcpxR mutant compared to the WT, whereas the expression levels of acrA in the CpxAR and PhoPQ modulated the expression of specific global regulatory factors. As presented in the C3 extended network (Fig. 2C), the global regulatory factors were key nodes in CpxAR-and PhoPQ-mediated regulation of efflux pump genes. qRT-PCR results showed that there were 2-to 4-fold increases in the expression levels of the marA, soxS, and ramA genes and a 4-fold decrease in the expression level of crp gene in NAL-treated cells, whereas no significant difference was observed in CAZtreated cells compared to untreated WT cells at 2 h (Fig. 5). When cells were treated at the early stationary stage, the expression level of ramA was significantly lower in the DcpxR mutant, but in the DphoP mutant, ramA expression was similar to or higher than that in the WT cells. In addition, the expression level of the crp gene in DphoP mutant was significantly (P , 0.001) increased, which had not been identified in previous studies under antibiotic conditions, indicating the negative regulation of PhoP on crp expression. Furthermore, the expression level of marA gene was increased and higher There existed a compensatory function between PhoP and CpxR. The expression levels of the phoP and cpxR genes in the WT and mutants were determined. Although the expression level of phoP showed no significant difference among DcpxR, NALtreated DcpxR (DcpxR1NAL) cells, and CAZ-treated DcpxR (DcpxR1CAZ) cells, a comparatively higher expression level of phoP was found in DcpxR cells than in WT cells (P , 0.05) (Fig. 6). Similarly, the expression level of cpxR in DphoP cells was significantly higher than those in WT cells. In addition, significantly higher expression levels of cpxR in DphoP1NAL or DphoP1CAZ cells than untreated DphoP cells were also observed (except for NAL treatment at 10 h). These data indicated that there was a compensation mechanism between phoP and cpxR functions, which might partially account for the antibiotic pressure fitness of S. Enteritidis.

DISCUSSION
In the present study, a large-scale mutational analysis was conducted on MDR S. Enteritidis to explore the role of TCSs in the development of antibiotic resistance. Our results revealed that 38.46% of the TCS pairs were involved in antibiotic resistance of S. Enteritidis. Among these TCSs, BarA/UvrY, NarQP, and ArcBA, previously known as regulators for metabolic control (37)(38)(39), were found to facilitate resistance development. Additionally, the absence of yehT and basR resulted in an increase in susceptibility of S. Enteritidis to ceftriaxone and colistin, respectively, in agreement with published studies where overexpression of yehT caused a 2-fold increase in MIC of b-lactams and mutation in PmrAB resulted in a decrease in resistance to colistin (19,40). However, in the present study, the mutants with deletion of baeR and ompR exhibited increased susceptibility to gentamicin and cefotaxime, respectively, whereas overexpression of baeR was previously reported to lead to increased resistance to ceftiofur and cefotaxime, and alteration of EnvZ/OmpR contributed to ertapenem and nalidixic acid resistance in E. coli (41)(42)(43). Thus, there were differences in the effects of TCS orthologs on bacterial resistance. The TCSs CpxAR, PhoPQ, and GlnGL were shown to play important roles in MDR of S. Enteritidis SJTUF12367, which was observed in multiple S. Enteritidis isolates, indicating the universal contribution of these TCSs to the MDR phenotype of S. Enteritidis serovars. It was previously reported that the absence of phoPQ led to attenuated expression of mltD1 and slt and increased b-lactam influxes in Stenotrophomonas maltophilia (44), and inactivation of CpxR resulted in higher susceptibility to aminoglycosides and b-lactams of S. Typhimurium (18). In our findings, phoP and cpxR mutants additionally exhibited higher susceptibility to nalidixic acid, ciprofloxacin, and ofloxacin with a drastic decrease (up to 8-fold) in MIC, suggesting a more extensive influence of these TCSs on antibiotic resistance. GlnGL (also called NtrBC) was a key regulator in response to nitrogen source and nitrogen availability via activation of expression of glnA, which encoded a glutamine synthetase (GS) (45). Recently, a few clues in susceptible strains showed that the absence of ntrBC in E. coli and glnA in Salmonella enterica serovar Typhi could increase their susceptibility to aminoglycosides and ciprofloxacin, respectively (46,47). In this study, a phenotype in S. Enteritidis glnG mutants of relative susceptibility to quinolones and aminoglycosides was observed, and the absence of glnG also led to an increase in susceptibility to cephalosporins and tetracycline, suggesting a greater influence of glnG on MDR S. Enteritidis than on susceptible strains. Overall, these TCSs were crucial for development of multidrug resistance in S. Enteritidis.
Gene network analysis is one of the reliable approaches that provide new insights into biological information of antimicrobial resistance mechanisms. In this work, the ARGs were placed into three clusters. The genes in cluster C1 (e.g., gyrA, rpoB, and rplC) were responsible for alteration in drug targets (48)(49)(50)(51)(52); however, there was only one single-base mutation in GyrA, D87G, found in the SJTUF12367 genome. The genes in cluster C2 (clsA, clsC and pgsA) were enriched in the phospholipid biosynthesis pathway (KW-0594) and responsible for synthesis of cardiolipin and phosphatidylglycerol synthesis, whose loss could be compensated for by other anionic phospholipids (53). The genes in cluster C3 were deemed essential for MDR phenotype, and the associations among PhoPQ, CpxAR as well as global regulators were found in the C3 extended network, which indicated a coupling of signaling of CpxAR or PhoPQ in modulating the global regulators and relevant genes of the efflux pump and OM porin. Unfortunately, no interaction links were found between glnGL and ARGs. Previous studies reported that glutamine was responsible for synthesis of a poly-a-L-glutamine layer associated with the cell wall, and inhibition of GS activity enhanced the susceptibility to penicillin in Streptococcus pneumoniae and methicillin in Staphylococcus aureus (54,55). In addition, the E. coli TCS GlnGL could govern transcription of relA/spoT genes and thereby led to elevated level of guanosine tetra-and pentaphosphate (ppGpp). In Vibrio cholerae, accumulation of ppGpp led to a suppressed central metabolism and iron transport system, resulting in reduced oxidative stress and increased antibiotic resistance (tetracycline, erythromycin, and chloramphenicol) (56,57). Altogether, we speculated that S. Enteritidis GlnGL could activate expression of GS and ppGpp, which were involved in production of cell envelope constituents and regulation of intracellular redox balance, therefore conferring antibiotic resistance.
The regulatory effects of CpxAR and PhoPQ on multiple ARGs were demonstrated in this study. Bacteria could survive antibiotic exposure by expelling antibiotics through efflux pumps and blocking OM porin-mediated antibiotic influx (58). The global regulators RamA, SoxS, and MarA could modulate expression of efflux pumps and OM porins in Salmonella (59,60). In this work, the absence of cpxR/phoP caused an increase in the expression level of OM porins and in membrane permeability as well as a decrease in expression level of specific efflux pumps and their regulatory factors, resulting in influx of a quantity of antibiotic molecules. Previous studies showed that the expression of OM porins STM1530 and OmpD was regulated by CpxAR to promote ceftriaxone resistance of S. Typhimurium (61). In E. coli, overproduction of CpxR was found to confer resistance to b-lactams in an acrB-free background (40). P. aeruginosa CpxAR was found to enhance quinolone resistance by activating expression of the mexAB-oprM efflux pump, while the absence of CpxR led to a Klebsiella pneumoniae phenotype susceptible to b-lactams and chloramphenicol (24,62). Therefore, the regulatory function of CpxAR on efflux pumps and OM porins was universal across different species. On the other hand, it was proposed that PhoPQ could activate the putative ABC transporters in P. aeruginosa to extrude intracellular tetracycline (63), and the MdtK pump could expel ciprofloxacin into the periplasmic space in E. coli (64). To our knowledge, there were no direct clues to the resistance regulatory function of PhoPQ in S. Enteritidis. This study clarified the correlation between PhoPQ and efflux pumps by demonstrating the upregulatory effect of PhoPQ on the transcription level of the ABC-type family and MATE family pump genes (macB and mdtK). In addition, PhoP was reported as a transcriptional activator of crp in Yersinia pestis (65) and the loss of crp in S. Typhimurium could enhance resistance to fluoroquinolones by reducing permeability and elevating efflux of fluoroquinolones (33). Given the increased level of expression of crp in DphoP cells, we proposed that the absence of PhoP could derepress expression of crp and affect the function of multiple ARGs. In any case, CpxAR and PhoPQ in S. Enteritidis could modulate expression of the ARGs that encoded OM porins, efflux pumps, and global regulators, conferring resistance to quinolones and cephalosporins.
Previous studies proposed that there was a feedback mechanism employed to regulate expression of AcrAB-TolC and other functional efflux pumps by RamA and MarA (30,60). Accordingly, a variety of compensatory pathways that improve fitness under the subinhibitory antibiotic pressure were presented in this study. Activation of EmrA pump in the absence of phoP or cpxR cells could be attributed to this feedback mechanism, which compensated for the loss of other pumps' efflux function. In addition, it has been demonstrated that CpxR might regulate the colistin susceptibility of S. Typhimurium through PmrAB and PhoPQ (66). In this work, CpxR and PhoP showed cross-interaction in gene interaction networks, and qRT-PCR analysis further confirmed a compensatory expression of these two regulator genes, indicating a partial functional redundancy between PhoPQ and CpxAR. Nevertheless, these fitness mechanisms were not sufficient to completely make up for the sensitive phenotype caused by phoP/cpxR deletion, despite the adaptation of the DphoP and DcpxR mutants to antibiotics.
In conclusion, three TCSs, CpxAR, PhoPQ, and GlnGL, were identified as important regulators for MDR formation in S. Enteritidis. Deficiency in each of these TCSs results in a significantly reduced resistance to at least three classes of antibiotics, and this change was universal in S. Enteritidis. CpxAR and PhoPQ conferred resistance to quinolones and cephalosporins by altering expression of the genes responsible for drug efflux and drug entry, as well as those that caused significant changes in the global regulators. These TCSs were therefore considered potential targets in synergistic antibacterial therapy of MDR S. Enteritidis. In-depth characterization of crucial ones (e.g., GlnGL) that operate through gene regulation to uncover multiple cellular pathways for MDR is ongoing in our laboratory.

MATERIALS AND METHODS
Bacterial strains. S. Enteritidis isolates (n = 7) were recovered from stool samples of patients with diarrhea during 2007 to 2017 in Shanghai and Henan, China, that were provided by the Shanghai Municipal Center for Disease Control and Prevention and reported in our recent papers (28,67). These isolates belonged to ST11 (the predominant ST in S. Enteritidis) and exhibited the frequent antimicrobial resistance profile of AMP-SXT-NAL-CAZ-CRO-CTX-FEP (see Table S1 in the supplemental material), among which S. Enteritidis SJTUF12367 had the widest drug resistance profile (ACSSuT-NAL-KAN-CAZ-CRO-CTX-ceftiofur [TIO]-FEP-fosfomycin [FOS]). These strains were cultivated in lysogeny broth (LB) or on LB agar supplemented with or without appropriate antibiotics.
Construction of gene deletion and complementation strains. The TCS elements of the SJTUF12367 genome were identified via HMMER software and the Pfam database (Table S3 and Table S4). TCS gene deletion and complementation were performed as described previously (68). Briefly, TCS genes were first replaced by an FRT site-flanked hph cassette, which was a hygromycin resistance gene that was used as the resistance selection marker to construct homologous recombinant fragments and conferred resistance to hygromycin of TCS deletion strains. The cassette was then eliminated via Flp-FRT recombination to obtain markerless in-frame indel mutants. For the construction of complementary strains, the full-length regulator protein gene and its ribosome binding site were cloned into the pBAD33 vector with an arabinose-inducible Role of TCSs in Drug Resistance of S. Enteritidis mSphere promoter and then transformed into the corresponding mutants. The resulting strains were confirmed by PCR and DNA sequencing. The plasmids and primers used are shown in Table S1 and Table S5, respectively. Antimicrobial susceptibility tests. Antimicrobial susceptibility testing was conducted on S. Enteritidis isolates using the standard agar dilution method recommended by the CLSI (69). The antibiotics used included third-and fourth-generation (broad-spectrum) cephalosporins (ceftazidime, ceftriaxone, cefotaxime, ceftiofur and cefepime) as well as nalidixic acid, ofloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, ampicillin, gentamicin, streptomycin, kanamycin, azithromycin, tetracycline, and chloramphenicol. All antibiotics were purchased from Sigma, Inc. E. coli ATCC 25922 was used as a quality control for the MIC value determination.
Construction of the gene interaction network. ARGs of the SJTUF12367 genome were collected from three databases, including the Pathway-systems Resource Investigation Center (PATRIC; https:// www.patricbrc.org/), Comprehensive Antibiotic Resistance Database (CARD; https://card.mcmaster.ca/), and National Database of Antibiotic Resistance Organisms (NDARO; https://www.ncbi.nlm.nih.gov/ pathogens/antimicrobial-resistance/) (70). The interacting partner data were curated from the STRING database with the medium confidence scores (more than 0.4). The gene interaction network was visualized by Cytoscape. The Cytoscape tools MCODE and NetworkAnalyzer were used for identification of the highly interacting nodes (clusters) and computing the topological parameters for networks, respectively.
Membrane perturbation assays. The 1-N-phenylnaphthylamine (NPN) uptake assay was performed to determine the outer membrane permeability (36). NPN was added to the culture containing 2 Â 10 6 CFU/mL S. Enteritidis wild-type and DcpxR/DphoP cells (final NPN concentration of 12.5 mM) and incubated for 15 min with various concentrations of antibiotics. The fluorescence emission intensity was recorded (l exc = 340 nm; l em = 420 nm; gap width, 1 mm) using an Infinite 200 PRO microplate reader (Tecan, Switzerland).
RNA isolation and qRT-PCR assays. The cultures of exponentially growing cells were equalized to an optical density at 600 nm (OD 600 ) value of 0.2. Volumes of 160-mL bacterial suspensions were then added to 3.84 mL LB, to which had been preadded subinhibitory concentrations of antibiotics to reach final concentrations of 32 mg/L (1/8 MIC) nalidixic acid (NAL) and 2 mg/L (1/16 MIC) ceftazidime (CAZ) (Fig. S4) (33). Samples were taken after incubation at 2 h and 8/10 h. RNA was extracted using TRIzol reagent (Invitrogen) as described by Huang et al. (48). Removal of residual genomic DNA and cDNA synthesis was conducted using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa). RNA integrity was confirmed by electrophoresis. The primers used are shown in Table S5. qRT-PCR was run in triplicate and amplified using the TB green Premix Ex Taq II reagent (TaKaRa). The relative gene transcriptional level was determined using the threshold cycle (2 2DDCT ) method (71) and converted into the log 2 value. The expression of 16S rRNA was used as an internal reference. qRT-PCR data analysis was undertaken by a two-way analysis of variance (ANOVA) and Holm-Sidak's multiple-comparison test via the Prism8.0.1 program, and all points were normalized relative to the untreated WT at 2 h.
Data availability. The complete genome sequence of S. Enteritidis SJTUF12367 containing a chromosome (GenBank accession no. CP003200) and two plasmids (GenBank accession no. CP041177 and CP041178) with abundant ARGs was deposited in NCBI (GenBank assembly accession no. GCA_004323915.1).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.   We declare no conflict of interest.