Novel Functionalized Spiro [Indoline-3,5'-pyrroline]-2,2'dione Derivatives: Synthesis, Characterization, Drug-Likeness, ADME, and Anticancer Potential.

A highly stereo-selective, one-pot, multicomponent method was chosen to synthesize the novel functionalized 1, 3-cycloaddition spirooxindoles (SOXs) (4a-4h). Synthesized SOXs were analyzed for their drug-likeness and ADME parameters and screened for their anticancer activity. Our molecular docking analysis revealed that among all derivatives of SOXs (4a-4h), 4a has a substantial binding affinity (∆G) -6.65, -6.55, -8.73, and -7.27 Kcal/mol with CD-44, EGFR, AKR1D1, and HER-2, respectively. A functional study demonstrated that SOX 4a has a substantial impact on human cancer cell phenotypes exhibiting abnormality in cytoplasmic and nuclear architecture as well as granule formation leading to cell death. SOX 4a treatment robustly induced reactive oxygen species (ROS) generation in cancer cells as observed by enhanced DCFH-DA signals. Overall, our results suggest that SOX (4a) targets CD-44, EGFR, AKR1D1, and HER-2 and induces ROS generation in cancer cells. We conclude that SOX (4a) could be explored as a potential chemotherapeutic molecule against various cancers in appropriate pre-clinical in vitro and in vivo model systems.

Genome-wide analysis of genes involved in efflux function and regulation within Escherichia coli and Salmonella enterica serovar Typhimurium.

The incidence of multidrug-resistant bacteria is increasing globally, with efflux pumps being a fundamental platform limiting drug access and synergizing with other mechanisms of resistance. Increased expression of efflux pumps is a key feature of most cells that are resistant to multiple antibiotics. Whilst expression of efflux genes can confer benefits, production of complex efflux systems is energetically costly and the expression of efflux is highly regulated, with cells balancing benefits against costs. This study used TraDIS-Xpress, a genome-wide transposon mutagenesis technology, to identify genes in Escherichia coli and Salmonella Typhimurium involved in drug efflux and its regulation. We exposed mutant libraries to the canonical efflux substrate acriflavine in the presence and absence of the efflux inhibitor phenylalanine-arginine ß-naphthylamide. Comparisons between conditions identified efflux-specific and drug-specific responses. Known efflux-associated genes were easily identified, including acrAB, tolC, marRA, ramRA and soxRS, confirming the specificity of the response. Further genes encoding cell envelope maintenance enzymes and products involved with stringent response activation, DNA housekeeping, respiration and glutathione biosynthesis were also identified as affecting efflux activity in both species. This demonstrates the deep relationship between efflux regulation and other cellular regulatory networks. We identified a conserved set of pathways crucial for efflux activity in these experimental conditions, which expands the list of genes known to impact on efflux efficacy. Responses in both species were similar and we propose that these common results represent a core set of genes likely to be relevant to efflux control across the Enterobacteriaceae.

New insights into KLFs and SOXs in cancer pathogenesis, stemness, and therapy.

Despite the development of cancer therapies, the success of most treatments has been impeded by drug resistance. The crucial role of tumor cell plasticity has emerged recently in cancer progression, cancer stemness and eventually drug resistance. Cell plasticity drives tumor cells to reversibly convert their cell identity, analogous to differentiation and dedifferentiation, to adapt to drug treatment. This phenotypical switch is driven by alteration of the transcriptome. Several pluripotent factors from the KLF and SOX families are closely associated with cancer pathogenesis and have been revealed to regulate tumor cell plasticity. In this review, we particularly summarize recent studies about KLF4, KLF5 and SOX factors in cancer development and evolution, focusing on their roles in cancer initiation, invasion, tumor hierarchy and heterogeneity, and lineage plasticity. In addition, we discuss the various regulation of these transcription factors and related cutting-edge drug development approaches that could be used to drug "undruggable" transcription factors, such as PROTAC and PPI targeting, for targeted cancer therapy. Advanced knowledge could pave the way for the development of novel drugs that target transcriptional regulation and could improve the outcome of cancer therapy.

Fusobacteria behaving badly: Masquerading strains of strictly anaerobic Escherichiacoli misidentified due to the deletion of the hemB gene.

Three strictly anaerobic strains of Escherichia coli were misidentified as Fusobacterium mortiferum, due to a deletion of the hemB gene which is involved in anaerobic respiration. An unusual antimicrobial susceptibility pattern sparked the further diagnostic strategies that eventually identified these strains as true anaerobic E. coli This phenomenon is more common than appreciated and can have an impact on clinical practice including persistent and relapsing infections.

HnRNPU-AS1 inhibits the proliferation, migration and invasion of HCC cells and induces autophagy through miR-556-3p/ miR-580-3p/SOCS6 axis.

BACKGROUND: Long non-coding RNAs have drawn increasing research interest in cancer biology. This study aims to investigate the function roles and the underlying mechanism of HnRNPU-AS1 in Hepatocellular carcinoma (HCC). METHODS: qRT-PCR was performed to detect the expression levels of HnRNPU-AS1, miR-556-3p, miR-580-3p in HCC tissues and cell lines. Western blot was used to determine protein levels of LC3-II, LC3-I, Beclin-1, P62, and SOCS6. Functional assays including CCK8 assay, colony formation assay, wound healing assay, Transwell assay were performed to evaluate the role of HnRNPU-AS1 in regulating the malignant phenotype of HCC cells. Dual luciferase reporter assay and RNA pull-down experiment were used to examined the RNA-RNA interaction. RESULTS: HnRNPU-AS1 expression was decreased in HCC tissues and cell lines, which was associated with poor prognosis in HCC patients. Overexpression of HnRNPU-AS1 could inhibit the proliferation, migration, invasion but promote autophagy in HCC cells. Two miRNAs (miR-556-3p and miR-580-3p) were identified as potential targets of HnRNPU-AS1 in lncBASE database, which were significantly upregulated in HCC tissues and cell lines. Cell experiments demonstrated the effects of HnRNPU-AS1 overexpression could be attenuated by miR-556-3p or miR-580-3p overexpression. We further revealed that SOX6 was the downstream target of HnRNPU-AS1/miR-556-3p or miR-580-3p axis. Xenograft mouse model validated the tumor-suppressor role of HnRNPU-AS1 overexpression in vivo. CONCLUSIONS: This study demonstrated the tumor suppressor function of HnRNPU-AS1 in HCC and identified the downstream molecules underlying its tumor suppressor function. Our results suggest that HnRNPU-AS1 suppresses HCC by targeting miR-556-3p and miR-580-3p/SOXS6 axis.

Antibacterial activity of honeys and potential synergism of honeys with antibiotics and alkaloid extract of Sophora alopecuroides plant against antibiotic-resistant Escherichia coli mutant.

OBJECTIVES: The increase in multidrug-resistant Escherichia coli strains with an overactivated AcrAB-TolC efflux pump has reduced the effectiveness of synthetic antibiotics, such as ciprofloxacin. The activity of this efflux pump can be reduced by using natural products. This study aimed to use a combination of ciprofloxacin, honey, and alkaloid extract of Sophora alopecuroides against an E. coli mutant with an overactivated AcrAB-TolC pump. MATERIALS AND METHODS: First the physicochemical properties, total alkaloid content, antioxidant activity, and the minimum inhibitory concentration (MIC) of three local honey samples: Konar (lotus), Avishan (Thyme), and Gavan (Astragalus) were evaluated. Then, the MIC of different combinations of honey, ciprofloxacin, and plant alkaloid extract and expression of acrA and soxS genes were carried out using the agar dilution method and quantitative RT- PCR methods. RESULTS: The net absorbance, total alkaloid content, and DPPH radical scavenging activity of Konar honey were significantly higher than those of Avishan and Gavan honeys (P<0.05). However, the MIC of lotus honey was nearly similar to other honey types, and all honey (30% w/v)-ciprofloxacin combinations decreased the viability of mutant more than ciprofloxacin alone. A synergistic interaction (FICI =0.48) was observed in triplex complex of ciprofloxacin (10 µg/ml), honey (20% w/v), and plant extract (1 mg/ml). A significant decrease (P<0.05) in the expression level of genes was seen in the presence of the triplex complex. CONCLUSION: It is concluded that the interaction between honey and plant alkaloid extract enhanced the anti-pump activity and reduced the oxidative stress response of the E. coli mutant.

Analysis of the Oxidative Stress Regulon Identifies soxS as a Genetic Target for Resistance Reversal in Multidrug-Resistant Klebsiella pneumoniae.

In bacteria, the defense system deployed to counter oxidative stress is orchestrated by three transcriptional factors, SoxS, SoxR, and OxyR. Although the regulon that these factors control is known in many bacteria, similar data are not available for Klebsiella pneumoniae. To address this data gap, oxidative stress was artificially induced in K. pneumoniae MGH78578 using paraquat and the corresponding oxidative stress regulon recorded using transcriptome sequencing (RNA-seq). The soxS gene was significantly induced during oxidative stress, and a knockout mutant was constructed to explore its functionality. The wild type and mutant were grown in the presence of paraquat and subjected to RNA-seq to elucidate the soxS regulon in K. pneumoniae MGH78578. Genes that are commonly regulated both in the oxidative stress and soxS regulons were identified and denoted as the oxidative SoxS regulon; these included a group of genes specifically regulated by SoxS. Efflux pump-encoding genes and global regulators were identified as part of this regulon. Consequently, the isogenic soxS mutant was found to exhibit a reduction in the minimum bactericidal concentration against tetracycline compared to that of the wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further evaluated using a tetraphenylphosphonium (TPP+) accumulation assay. The soxS mutant was also susceptible to tetracycline in vivo in a zebrafish embryo model. We conclude that the soxS gene could be considered a genetic target against which an inhibitor could be developed and used in combinatorial therapy to combat infections associated with multidrug-resistant K. pneumoniae. IMPORTANCE Antimicrobial resistance is a global health challenge. Few new antibiotics have been developed for use over the years, and preserving the efficacy of existing compounds is an important step to protect public health. This paper describes a study that examines the effects of exogenously induced oxidative stress on K. pneumoniae and uncovers a target that could be useful to harness as a strategy to mitigate resistance.

Cell Lysis Directed by SulA in Response to DNA Damage in Escherichia coli.

The SOS response is induced upon DNA damage and the inhibition of Z ring formation by the product of the sulA gene, which is one of the LexA-regulated genes, allows time for repair of damaged DNA. On the other hand, severely DNA-damaged cells are eliminated from cell populations. Overexpression of sulA leads to cell lysis, suggesting SulA eliminates cells with unrepaired damaged DNA. Transcriptome analysis revealed that overexpression of sulA leads to up-regulation of numerous genes, including soxS. Deletion of soxS markedly reduced the extent of cell lysis by sulA overexpression and soxS overexpression alone led to cell lysis. Further experiments on the SoxS regulon suggested that LpxC is a main player downstream from SoxS. These findings suggested the SulA-dependent cell lysis (SDCL) cascade as follows: SulA→SoxS→LpxC. Other tests showed that the SDCL cascade pathway does not overlap with the apoptosis-like and mazEF cell death pathways.

Silver Nanoparticles Induce a Triclosan-Like Antibacterial Action Mechanism in Multi-Drug Resistant Klebsiella pneumoniae.

Infections associated with antimicrobial-resistant bacteria now represent a significant threat to human health using conventional therapy, necessitating the development of alternate and more effective antibacterial compounds. Silver nanoparticles (Ag NPs) have been proposed as potential antimicrobial agents to combat infections. A complete understanding of their antimicrobial activity is required before these molecules can be used in therapy. Lysozyme coated Ag NPs were synthesized and characterized by TEM-EDS, XRD, UV-vis, FTIR spectroscopy, zeta potential, and oxidative potential assay. Biochemical assays and deep level transcriptional analysis using RNA sequencing were used to decipher how Ag NPs exert their antibacterial action against multi-drug resistant Klebsiella pneumoniae MGH78578. RNAseq data revealed that Ag NPs induced a triclosan-like bactericidal mechanism responsible for the inhibition of the type II fatty acid biosynthesis. Additionally, released Ag+ generated oxidative stress both extra- and intracellularly in K. pneumoniae. The data showed that triclosan-like activity and oxidative stress cumulatively underpinned the antibacterial activity of Ag NPs. This result was confirmed by the analysis of the bactericidal effect of Ag NPs against the isogenic K. pneumoniae MGH78578 ΔsoxS mutant, which exhibits a compromised oxidative stress response compared to the wild type. Silver nanoparticles induce a triclosan-like antibacterial action mechanism in multi-drug resistant K. pneumoniae. This study extends our understanding of anti-Klebsiella mechanisms associated with exposure to Ag NPs. This allowed us to model how bacteria might develop resistance against silver nanoparticles, should the latter be used in therapy.

Transcriptional response of mar, sox and rob regulon against concentration gradient carbapenem stress within Escherichia coli isolated from hospital acquired infection.

OBJECTIVE: The present study was carried out to investigate the transcriptional response of marA (Multiple antibiotic resistance A gene), soxS (Superoxide S gene) and rob (Right-origin-binding gene) under carbapenem stress. RESULTS: 12 isolates were found over-expressing AcrAB-TolC efflux pump system and showed reduced expression of OmpF (Outer membrane porin) gene were selected for further study. Among them, over expression of marA and rob was observed in 7 isolates. Increasing pattern of expression of marA and rob against meropenem was observed. The clones of marA and rob showed reduced susceptibility towards carbapenems.

SoxS is a positive regulator of key pathogenesis genes and promotes intracellular replication and virulence of Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important intracellular pathogen, causing gastroenteritis or severe systemic infection in a variety of hosts. During infection, S. Typhimurium must survive and replicate in host macrophages, which produce abundant oxidative compounds. SoxRS regulon is a well-known regulator that is activated in response to oxidative stress and promotes bacterial tolerance to oxidants in E. coli. However, the global regulatory function of SoxS in S. Typhimurium remains poorly characterized. Here, we used an RNA sequencing-based approach to investigate the role of SoxS in the expression of S. Typhimurium virulence genes. Besides the downregulation of genes related to resistance to oxidative stress, we found that in a soxS deletion mutant the expression of Salmonella pathogenicity island (SPI)-2 genes, which are crucial for replication within macrophages, was significantly repressed. Moreover, immunofluorescence and mice infection experiments showed that soxS deletion inhibited replication in macrophages and decreased virulence upon intraperitoneal inoculation in mice, respectively. Collectively, our findings demonstrate that SoxS is a positive regulator of SPI-2 genes and, therefore, plays a crucial role in S. Typhimurium intracellular replication and virulence.

OmpW is positively regulated by iron via Fur, and negatively regulated by SoxS contribution to oxidative stress resistance in Escherichia coli.

Iron plays a central role at the interface of pathogen and host. The ability to sequester iron from a host not only reduces host immune defenses but also promotes pathogen virulence, leading to the occurrence of infectious disease. Recently, outer membrane protein OmpW was shown to protect bacteria against harsh environmental conditions and to play a role in infectious disease. The expression of this versatile protein is controlled by iron, but the underlying mechanism of iron regulation has not been elucidated. In this study, the relation between OmpW expression and iron was investigated. Our results demonstrated that expression of OmpW is responsive to iron. Iron uptake analysis showed that an ompW mutant strain has a strong requirement for iron as compared to wild type and the ompW complemented strain. Moreover, ferric uptake regulation protein Fur, an iron binding transcriptional factor, was downregulated under iron limitation conditions and had a similar expression profile to OmpW in the presence or absence of iron. Based on these results, we suggest that iron regulates OmpW by binding to Fur. Furthermore, SoxS, a transcriptional factor involved in oxidative stress, was found to negatively regulate OmpW. We found that downregulating or knocking out OmpW results in bacterial resistance to oxidative stress. These findings provide new insight into the regulation of OmpW expression by iron, and may represent a new mechanism contributing to iron-mediated infectious disease.

Impact of Acceptor Fluorination on the Performance of All-Polymer Solar Cells.

Here, we systematically study the effect of fluorination on the performance of all-polymer solar cells by employing a naphthalene diimide (NDI)-based polymer acceptor with thiophene-flanked phenyl co-monomer. Fluorination of the phenyl co-monomer with either two or four fluorine units is used to create a series of acceptor polymers with either no fluorination (PNDITPhT), bifluorination (PNDITF2T), or tetrafluorination (PNDITF4T). In blends with the donor polymer PTB7-Th, fluorination results in an increase in power conversion efficiency from 3.1 to 4.6% despite a decrease in open-circuit voltage from 0.86 V (unfluorinated) to 0.78 V (tetrafluorinated). Countering this decrease in open-circuit voltage is an increase in short-circuit current from 7.7 to 11.7 mA/cm2 as well as an increase in fill factor from 0.45 to 0.53. The origin of the improvement in performance with fluorination is explored using a combination of morphological, photophysical, and charge-transport studies. Interestingly, fluorination is found not to affect the ultrafast charge-generation kinetics, but instead is found to improve charge-collection yield subsequent to charge generation, linked to improved electron mobility and improved phase separation. Fluorination also leads to improved light absorption, with the blue-shifted absorption profile of the fluorinated polymers complementing the absorption profile of the low-band gap PTB7-Th.

pqiABC and yebST, Putative mce Operons of Escherichia coli, Encode Transport Pathways and Contribute to Membrane Integrity.

The membranes of single-cell organisms are crucial as the first line of defense. The outer membrane of Gram-negative bacteria is an asymmetric bilayer in which lipopolysaccharides (LPSs) and phospholipids are localized in the outer and inner leaflet, respectively. This asymmetry is important for membrane integrity. In Escherichia coli, the Mla transport pathway maintains this asymmetry by removing phospholipids from the outer leaflet. The MlaD component of this system is a mammalian cell entry (MCE) domain protein, and E. coli has two other MCE domain proteins of unknown function (PqiB and YebT). Here, we show that these two proteins are components of novel transport pathways that contribute to membrane integrity. The pqiAB operon is regulated by SoxS and RpoS. The yebST operon contains pqiAB homologues. Here, we found a third member of the pqi operon, ymbA (pqiC). A PqiB-PqiC complex bridges the inner and the outer membrane, and in other bacteria, pqiBC genes are located in operons together with transporter proteins. We show here that simultaneous deletion of pqiABC and yebST operons in an Δmla background rendered cells more sensitive to SDS-EDTA, and the SDS-EDTA sensitivity of mla mutants was rescued by additional copies of pqiABC We also found that the yebST operon was induced by a defect in LPS molecules. In conclusion, PqiABC and YebST are novel transport pathways related to the Mla transport pathway and important for membrane integrity. IMPORTANCE: Membranes of bacteria are crucial for stress resistance. The composition of the E. coli outer membrane is asymmetric, with asymmetry maintained by the Mla ABC transport pathway. We propose that the stress-inducible pqiABC operon and homologous yebST operon, both of previously unknown function, encode transport pathway proteins related to the Mla transport pathway. Deletion of these operons rendered cells more sensitive to membrane stress, and additional copies of pqiABC suppressed the SDS-EDTA sensitivity of mla mutant strains. We found that yebS'-'lacZ fusion was activated in mutant strains with defective LPS molecules.

Evaluation of Oxidative Metabolism in Leukocytes during Phagocytosis of Escherichia coli Carrying Genetic Constructs soxS::lux or katG::lux.

We studied ROS generation by human peripheral blood monocytes and granulocytes during phagocytosis of Escherichia coli soxS::lux or katG::lux responding by luminescence (bioluminescence) to the development of oxidative stress. Initially high sensitivity of the bioluminescent reaction of E. coli katG::lux strain to the effects of model ROS (KO2 and H2O2) and pronounced induction of luminescence upon contact with granulocytes, whereas E. coli soxS::lux demonstrated less pronounced reaction to chemical oxidants and bioluminescence was observed primarily upon contact with monocytes. A correlation was found between quantitative characteristics of E. coli katG::lux bioluminescence and luminol-dependent chemiluminescence of leukocytes in some patients, but no dependence of this kind was noted for E. coli soxS::lux. The results can provide experimental substantiation of a new approach for evaluation of ROS production by leukocytes during phagocytosis and choosing the optimal object for these studies.

A novel alanine to serine substitution mutation in SoxS induces overexpression of efflux pumps and contributes to multidrug resistance in clinical Escherichia coli isolates.

OBJECTIVES: The purpose of this study was to describe a putative role for a novel soxS mutation in contributing to multiple-antibiotic resistance in canine fluoroquinolone-associated MDR (FQ-MDR) Escherichia coli. This soxS mutation was discovered in canine faecal E. coli isolates during a study investigating the effect of oral fluoroquinolone administration on faecal E. coli in healthy dogs. METHODS: We determined via quantitative real-time RT-PCR that both soxS and acrB were overexpressed in the clinical soxS Ala-12→Ser (soxS(A12S)) mutants and this may account for their FQ-MDR phenotype. We validated the FQ-MDR phenotype of the clinical isolates by reconstructing the WT and the soxS(A12S) mutation in the E. coli soxS null mutant JW4023 (soxS::kn) via allelic exchange. RESULTS: The JW4023 soxS(A12S) derivative showed an increase in MICs of ciprofloxacin, enrofloxacin and chloramphenicol compared with the JW4023 derivative in which the WT soxS had been restored. The soxS and acrB genes were overexpressed in the JW4023 soxS(A12S) mutant compared with JW4023 with WT soxS. A similar overexpression of efflux pump genes and an increase in antibiotic resistance were observed upon stimulation with paraquat to resemble the phenotype of the clinical soxS(A12S) isolates. CONCLUSIONS: Our data suggest that the soxS(A12S) substitution mutation is selected in clinical isolates when dogs are exposed to a fluoroquinolone and that this mutation contributes to the FQ-MDR phenotype of E. coli isolates.

Importance of domain purity and molecular packing in efficient solution-processed small-molecule solar cells.

Connections are delineated between solar-cell performance, charge-carrier mobilities, and morphology in a highperformance molecular solar cell. The observations show that maximizing the relative phase purity and structural order while simultaneously limiting the domain size may be essential for achieving optimal solar-cell performances in solution-processed small-molecule solar cells .

Interplay of solvent additive concentration and active layer thickness on the performance of small molecule solar cells.

A relationship between solvent additive concentration and active layer thickness in small-molecule solar cells is investigated. Specifically, the additive concentration must scale with the amount of semiconductor material and not as absolute concentration in solution. Devices with a wide range of active layers with thickness up to 200 nm can readily achieve efficiencies close to 6% when the right concentration of additive is used.

Regulation of acrAB expression by cellular metabolites in Escherichia coli.

OBJECTIVES: Multidrug efflux pumps mediate resistance to antibiotics and other toxic compounds. We studied the role of AcrAB-TolC, the main efflux pump in Escherichia coli, in regulating gene expression. METHODS: Deletion mutants, an acrABp-lacZ fusion and reverse transcription-real-time quantitative PCR experiments were used to study the role of AcrAB-TolC and metabolism in regulating gene expression of the acrAB operon and its transcriptional regulators. RESULTS: Deletion of the acrB gene increased the expression of the acrAB operon. A similar induction of acrAB was found when acrA or tolC was deleted, and when the pump function was inhibited using phenylalanine-arginine-ß-naphthylamide. The induction of acrAB in the ΔacrB strain was totally (AcrR or SoxS) or partially (SoxR or MarA) prevented when the genes for these acrAB regulators were also deleted. The expression of soxS and marA, but not of acrR, was increased in the ΔacrB strain, which also showed altered expression of many other genes related to different cellular processes, including motility. Deletion of the metabolic genes entA and entE (enterobactin biosysnthesis), glpX (gluconeogenesis), cysH (cysteine biosynthesis) and purA (purine biosynthesis) also prevented activation of the acrAB promoter in the ΔacrB strain. Addition of the enterobactin biosynthesis intermediate metabolite 2,3-dihydroxybenzoate induced the expression of acrAB. CONCLUSIONS: These results together suggest a model in which the AcrAB-TolC pump effluxes cellular metabolites that are toxic and/or have a signalling role. If the pump is inactivated or inhibited, these metabolites would accumulate, inactivating AcrR and/or up-regulating soxS and marA expression, ultimately triggering the up-regulation of acrAB expression to restore homeostasis.

Effect of Antimicrobial Exposure on AcrAB Expression in Salmonella enterica Subspecies enterica Serovar Choleraesuis.

Understanding the impact of antimicrobial use on the emergence of resistant bacteria is imperative to prevent its emergence. For instance, activation of the AcrAB efflux pumps is responsible for the emergence of antimicrobial-resistant Salmonella strains. Here, we examined the expression levels of acrB and its multiple regulator genes (RamA, SoxS, MarA, and Rob) in 17 field isolates of S. Choleraesuis by using quantitative PCR methods. The expression of acrB increased in eight of the field isolates (P < 0.05). The expression of acrB was associated with that of ramA in one isolate, soxS in one isolate, and both these genes in six isolates. Thereafter, to examine the effect of selected antimicrobials (enrofloxacin, ampicillin, oxytetracycline, kanamycin, and spectinomycin) on the expression of acrB and its regulator genes, mutants derived from five isolates of S. Choleraesuis were selected by culture on antimicrobial-containing plates. The expression of acrB and ramA was higher in the mutants selected using enrofloxacin (3.3-6.3- and 24.5-37.7-fold, respectively), ampicillin (1.8-7.7- and 16.1-55.9-fold, respectively), oxytetracycline (1.7-3.3- and 3.2-31.1-fold, respectively), and kanamycin (1.6-2.2- and 5.6-26.4-fold, respectively), which are AcrAB substrates, than in each of the parental strains (P < 0.05). In contrast, in AcrAB substrate-selected mutants, the expression of soxS, marA, and rob remained similar to that in parental strains. Of the four antimicrobials, the level of ramA expression was significantly higher in the enrofloxacin- and ampicillin-selected mutants than in the oxytetracycline- and kanamycin-selected mutants (P < 0.05), whereas the expression levels of acrB and multiple regulator genes in spectinomycin-selected mutants were similar to those in each parental strain. These data suggest that exposure to antimicrobials that are AcrAB substrates enhance the activation of the AcrAB efflux pump via RamA, but not via SoxS, MarA, or Rob in S. Choleraesuis.