Using random forest to predict antimicrobial minimum inhibitory concentrations of nontyphoidal Salmonella in Taiwan.

Antimicrobial resistance (AMR) is a global health issue and surveillance of AMR can be useful for understanding AMR trends and planning intervention strategies. Salmonella, widely distributed in food-producing animals, has been considered the first priority for inclusion in the AMR surveillance program by the World Health Organization (WHO). Recent advances in rapid and affordable whole-genome sequencing (WGS) techniques lead to the emergence of WGS as a one-stop test to predict the antimicrobial susceptibility. Since the variation of sequencing and minimum inhibitory concentration (MIC) measurement methods could result in different results, this study aimed to develop WGS-based random forest models for predicting MIC values of 24 drugs using data generated from the same laboratories in Taiwan. The WGS data have been transformed as a feature vector of 10-mers for machine learning. Based on rigorous validation and independent tests, a good performance was obtained with an average mean absolute error (MAE) less than 1 for both validation and independent test. Feature selection was then applied to identify top-ranked 10-mers that can further improve the prediction performance. For surveillance purposes, the genome sequence-based machine learning methods could be utilized to monitor the difference between predicted and experimental MIC, where a large difference might be worthy of investigation on the emerging genomic determinants.

Effects of L-arabinose efflux on λ Red recombination-mediated gene knockout in multiple-antimicrobial-resistant Salmonella enterica serovar Choleraesuis.

In this study, six swine-derived multiple-antimicrobial-resistant (MAR) strains of Salmonella Choleraesuis (S. Choleraesuis) were demonstrated to possess higher efflux pump activity than the wild-type (WT). L-Arabinose, a common inducer for gene expression, modulated S. Choleraesuis efflux pump activity in a dose-dependent manner. At low L-arabinose concentrations, increasing L-arabinose led to a corresponding increase in fluorophore efflux, while at higher L-arabinose concentrations, increasing L-arabinose decreased fluorophore efflux activity. The WT S. Choleraesuis that lacks TolC (ΔtolC), an efflux protein associated with bacterial antibiotic resistance and virulence, was demonstrated to possess a significantly reduced ability to extrude L-arabinose. Further, due to the rapid export of L-arabinose, an efficient method for recombination-mediated gene knockout, the L-arabinose-inducible bacteriophage λ Red recombinase system, has a reduced recombination frequency (~ 12.5%) in clinically isolated MAR Salmonella strains. An increased recombination frequency (up to 60%) can be achieved using a higher concentration of L-arabinose (fivefold) for genetic manipulation and functional analysis for MAR Salmonella using the λ Red system. The study suggests that L-arabinose serves not only as an inducer of the TolC-dependent efflux system but also acts as a competitive substrate of the efflux system. In addition, understanding the TolC-dependent efflux of L-arabinose should facilitate the optimization of L-arabinose induction in strains with high efflux activity.

Regulation of the G2-M cell cycle progression by the ERK5-NFkappaB signaling pathway.

Elucidation of mechanisms regulating cell cycle progression is of fundamental importance for cell and cancer biology. Although several genes and signaling pathways are implicated in G1-S regulation, less is known regarding the mechanisms controlling cell cycle progression through G2 and M phases. We report that extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinases, is activated at G2-M and required for timely mitotic entry. Stimulation of ERK5 activated nuclear factor kappaB (NFkappaB) through ribosomal S6 kinase 2 (RSK2)-mediated phosphorylation and degradation of IkappaB. Furthermore, selective inhibition of NFkappaB at G2-M phases substantially delayed mitotic entry and inhibited transcription of G2-M-specific genes, including cyclin B1, cyclin B2, Plk-1, and cdc25B. Moreover, inhibition of NFkappaB at G2-M diminished mitosis induced by constitutive activation of ERK5, providing a direct link between ERK5, NFkappaB, and regulation of G2-M progression. We conclude that a novel ERK5-NFkappaB signaling pathway plays a key role in regulation of the G2-M progression.

Immunostimulation of sugar cane extract on neutrophils to Salmonella typhimurium infection in mice.

The aim of this study was to evaluate the immunomodulatory effects of sugar cane extract (SCE) on the biological activities of neutrophils in mice. Six-week-old BALB/c mice were fed 1250 mg/kg of SCE once. The generation, migration and biological functions of neutrophils and the survival rates of the mice in response to Salmonella typhimurium infection were evaluated. The results show that the numbers of both bone marrow cells and neutrophils were significantly increased in response to SCE administration (p < 0.05) compared with controls. The migration, phagocytosis and H2O2 generation of neutrophils were all significantly enhanced in SCE-treated mice (p < 0.05). After challenge with S. typhimurium (lethal dose, 50% (LD50), SCE-treated mice had a 19.2% higher survival rate and milder hepatic lesions than the controls. Additionally, fewer invasive bacteria were recovered from the spleens of SCE-treated mice. In conclusion, our results suggest that SCE has a positive regulatory effect on the biological function of mouse neutrophils that may increase host resistance against bacterial infections.

Mechanisms underlying Actinobacillus pleuropneumoniae exotoxin ApxI induced expression of IL-1ß, IL-8 and TNF-α in porcine alveolar macrophages.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes fibrino-hemorrhagic necrotizing pleuropneumonia in pigs. Production of proinflammatory mediators in the lungs is an important feature of A. pleuropneumoniae infection. However, bacterial components other than lipopolysaccharide involved in this process remain unidentified. The goals of this study were to determine the role of A. pleuropneumoniae exotoxin ApxI in cytokine induction and to delineate the underlying mechanisms. Using real-time quantitative PCR analysis, we found native ApxI stimulated porcine alveolar macrophages (PAMs) to transcribe mRNAs of IL-1ß, IL-8 and TNF-α in a concentration- and time-dependent manner. Heat-inactivation or pre-incubation of ApxI with a neutralizing antiserum attenuated ApxI bioactivity to induce cytokine gene expression. The secretion of IL-1ß, IL-8 and TNF-α protein from PAMs stimulated with ApxI was also confirmed by quantitative ELISA. In delineating the underlying signaling pathways contributing to cytokine expression, we observed mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK) were activated upon ApxI stimulation. Administration of an inhibitor specific to p38 or JNK resulted in varying degrees of attenuation on ApxI-induced cytokine expression, suggesting the differential regulatory roles of p38 and JNK in IL-1ß, IL-8 and TNF-α production. Further, pre-incubation of PAMs with a CD18-blocking antibody prior to ApxI stimulation significantly reduced the activation of p38 and JNK, and subsequent expression of IL-1ß, IL-8 or TNF-α gene, indicating a pivotal role of ß2 integrins in the ApxI-mediated effect. Collectively, this study demonstrated ApxI induces gene expression of IL-1ß, IL-8 and TNF-α in PAMs that involves ß2 integrins and downstream MAPKs.

Actinobacillus pleuropneumoniae exotoxin ApxI induces cell death via attenuation of FAK through LFA-1.

ApxI exotoxin is an important virulence factor derived from Actinobacillus pleuropneumoniae that causes pleuropneumonia in swine. Here, we investigate the role of lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), a member of the ß2 integrin family, and the involvement of the integrin signaling molecules focal adhesion kinase (FAK) and Akt in ApxI cytotoxicity. Using Western blot analysis, we found that ApxI downregulated the activity of FAK and Akt in porcine alveolar macrophages (AMs). Preincubation of porcine AMs with an antibody specific for porcine CD18 reduced ApxI-induced cytotoxicity as measured by a lactate dehydrogenase release assay and decreased ApxI-induced FAK and Akt attenuation, as shown by Western blot analysis. Pretreatment with the chemical compounds PMA and SC79, which activate FAK and Akt, respectively, failed to overcome the ApxI-induced attenuation of FAK and Akt and death of porcine AMs. Notably, the transfection experiments revealed that ectopic expression of porcine LFA-1 (pLFA-1) conferred susceptibility to ApxI in ApxI-insensitive cell lines, including human embryonic kidney 293T cells and FAK-deficient mouse embryonic fibroblasts (MEFs). Furthermore, ectopic expression of FAK significantly reduced ApxI cytotoxicity in pLFA-1-cotransfected FAK-deficient MEFs. These findings show for the first time that pLFA-1 renders cells susceptible to ApxI and ApxI-mediated attenuation of FAK activity via CD18, thereby contributing to subsequent cell death.

Expression and immunological studies of classical swine fever virus glycoprotein E2 in the bi-cistronic baculovirus/larvae expression system.

To develop an economical, easy technique for producing recombinant E2 glycoprotein (rE2) of classical swine fever virus (CSFV) as a candidate immunogen, a bi-cistronic baculovirus/larvae expression vector was constructed using p10 promoter, an internal ribosome entry site, and the gfp gene. Trichoplusia ni larvae were successfully infected with the occluded recombinant baculovirus via feed, and the characteristics of rE2 were confirmed by immunoblot and glycosylation stain. rE2 at a concentration of 0.6-0.8 mg/ml without degradation was obtained from hemolymphs of infected larvae that emitted high levels of green fluorescence. Immunization assays indicated that mice and piglets immunized with rE2-containing hemolymph elicited high titers of anti-CSFV E2 antibodies with virus-neutralizing activity. This is the first study to indicate that baculovirus/T. ni larvae-expressed rE2 can be served as a vaccine candidate. This system provides an economical alternative for the production of vaccine components in the veterinary industry.

Suppurative meningitis in a 7-day-old Formosan sambar deer (Cervus unicolor swinhoei) caused by Escherichia coli.

This article describes the clinical and pathological features of an orphan 7-day-old, male Formosan sambar fawn that was hospitalized for treatment of weakness. The fawn had been deprived of colostrum and developed suppurative meningitis that was attributed to Escherichia coli.

Actinobacillus pleuropneumoniae serotype 10 derived ApxI induces apoptosis in porcine alveolar macrophages.

Actinobacillus pleuropneumoniae (AP) is the causative agent of swine pleuropneumonia, a fibrinous, exudative, hemorrhagic, necrotizing pleuropneumonia affecting all ages of pigs. Actinobacillus pleuropneumoniae exotoxins (Apx) are one of the major virulence factors of AP. Due to the complex nature of Apx toxins produced by AP, little is known regarding the interactions of individual species of Apx toxin with target cells. The objective of this study was to examine whether AP serotype 10-derived exotoxin, ApxI, caused apoptosis in porcine alveolar macrophages (PAMs) and to delineate the underlying signaling pathways. Isolated PAMs were stimulated with different concentrations of native ApxI and monitored for apoptosis using Hoechst staining, TUNEL, and DNA laddering assays. The ApxI-stimulated PAMs exhibited typical morphological features of apoptosis, including condensation of chromatin, formation of apoptotic bodies and DNA laddering. ApxI-induced apoptosis in a concentration- and time-dependent manner. Furthermore, to delineate the signaling events involved in ApxI-induced apoptosis, it was observed that caspase 3 was activated in ApxI-stimulated PAMs. Ablation of caspase 3 activity via specific inhibitors protected PAMs from apoptosis by ApxI. This study is the first to demonstrate that native ApxI causes apoptosis in PAMs at low concentrations and that these apoptotic events are mediated via a caspase 3-dependent pathway. These findings suggest a role of ApxI in AP infection as it might impair the host defense system through the induction of apoptosis in PAMs.

Analysis of ciprofloxacin-resistant Salmonella strains from swine, chicken, and their carcasses in Taiwan and detection of parC resistance mutations by a mismatch amplification mutation assay PCR.

One hundred thirty-three Salmonella strains isolated from the viscera of swine, chicken, and carcasses of swine and chicken in Taiwan from 2004 to 2006 were identified to serotype level and analyzed for their susceptibility to ciprofloxacin. In total, 76 (57%) strains of the Salmonella isolates exhibited high-level resistance to ciprofloxacin, having MICs ranging from 16 to 64 microg/ml. DNA sequence analysis revealed that mutations in the quinolone resistance-determining regions of gyrA (Ser83Phe, Asp87Gly or Asp87Asn), parC (Ser80Arg, or Ser80Ile or Glu84Lys), and parE (Ser458Pro) genes were associated with the Salmonella strains that demonstrated resistance to ciprofloxacin. A mismatch amplification mutation assay (MAMA)-PCR was developed to identify mutations in parC at codons 80 and 84. Specific PCR products were only recovered from ciprofloxacin-resistant Salmonella strains but not from the susceptible strains. MAMA-PCR targeting the mutations in parC correlated with what DNA sequencing revealed. In conclusion, monitoring ciprofloxacin-resistant Salmonella from animal sources should be performed on a regular basis. MAMA-PCR targeting parC could provide a fast method for those laboratories interested in quickly characterizing the resistance profile and with little access to DNA sequencing facilities.

Effect of immunostimulation by detoxified E. coli lipopolysaccharide combined with inactivated Propionibacterium granulosum cells on porcine immunity.

The aim of this experiment was to evaluate the immunomodulating activities of inactivated Propionibacterium granulosum cell walls and E. coli lipopolysaccharide (PG/LPS) on porcine immunity. Piglets were intramuscularly administered PG/LPS (1 ml/10 kg body weight) once or twice. The function of natural killer cells, lymphocytes and neutrophils and the adjuvant effect on antibody induction by attenuated classical swine fever virus (CSFV) and inactivated Mycoplasma hyopneumoniae vaccination were evaluated. The results showed that the cytotoxicity of natural killer cells and proliferation of lymphocytes in response to mitogen stimulation were significantly enhanced (P<0.05) in those pigs receiving PG/LPS injection compared with the controls. However, there was no significant effect on the phagocytic activity of neutrophils (P>0.05). PG/LPS also displayed adjuvant effects with CSFV and Mycoplasma hyopneumoniae vaccines. Moreover, pigs receiving two injections of PG/LPS showed a 20.8% growth enhancement compared with untreated pigs. Thus, PG/LPS caused positive immunoregulation of porcine innate immune system effectors, non-specific activation of lymphocytes and antibody production.

Spontaneous rhabdomyosarcoma in a young Sprague-Dawley rat.

An eight-week-old male Sprague-Dawley (SD) rat showed signs of emaciation, and masses were found in the subcutis around the cervical and thoracic regions. At necropsy, a multilobular mass, 2.2 x 1.8 x 2.0 cm in size, had grown from the left neck into the thoracic cavity. On a cutting surface, masses were firm and whitish to tan, with necrotic and hemorrhagic plaques. Microscopically, masses were composed of multiple nodules of tumor cells that were incompletely encapsulated with fibrous connective tissue. The tumor cells exhibited varied forms, from spindle to globoid shapes with minimal to abundant eosinophilic cytoplasm, and appeared as large, multinucleated cells; many of the tumor cells were vacuolated. Histochemistry results revealed that tumor cells exhibited some cross-striation in the cytoplasm using PTAH staining. There were some multinuclear tumor cells with vacuoles located around the nuclei, and these vacuoles showed reddish staining by the periodic acid Schiff (PAS) method. Immunohistochemical staining also expressed intense granular cytoplasmic staining for desmin and myoglobin, with highly positive staining for PCNA, whereas alpha-smooth muscle actin (alpha-SMA) was negative. Based on the pathology results, a spontaneous rhabdomyosarcoma with pleomorphic type was diagnosed in a young SD rat.

Molecular cloning, expression, and functional characterization of the ß-agarase AgaB-4 from Paenibacillus agarexedens.

In this study, a ß-agarase gene, agaB-4, was isolated for the first time from the agar-degrading bacterium Paenibacillus agarexedens BCRC 17346 by using next-generation sequencing. agaB-4 consists of 2652 bp and encodes an 883-amino acid protein with an 18-amino acid signal peptide. agaB-4 without the signal peptide DNA was cloned and expressed in Escherichia coli BL21(DE3). His-tagged recombinant AgaB-4 (rAgaB-4) was purified from the soluble fraction of E. coli cell lysate through immobilized metal ion affinity chromatography. The optimal temperature and pH of rAgaB-4 were 55 °C and 6.0, respectively. The results of a substrate specificity test showed that rAgaB-4 could degrade agar, high-melting point agarose, and low-melting point agarose. The Vmax and Km of rAgaB-4 for low-melting point agarose were 183.45 U/mg and 3.60 mg/mL versus 874.61 U/mg and 9.29 mg/mL for high-melting point agarose, respectively. The main products of agar and agarose hydrolysis by rAgaB-4 were confirmed to be neoagarotetraose. Purified rAgaB-4 can be used in the recovery of DNA from agarose gels and has potential application in agar degradation for the production of neoagarotetraose.

Basic fibroblast growth factor protects against rotenone-induced dopaminergic cell death through activation of extracellular signal-regulated kinases 1/2 and phosphatidylinositol-3 kinase pathways.

Administration of rotenone to rats reproduces many features of Parkinson's disease, including dopaminergic neuron degeneration, and provides a useful model to study the pathogenesis of Parkinson's disease. However, the cell death mechanisms induced by rotenone and potential neuroprotective mechanisms against rotenone are not well defined. Here we report that rotenone-induced apoptosis in human dopaminergic SH-SY5Y cells is attenuated by pretreatment with several growth factors, most notably basic fibroblast growth factor (bFGF). bFGF activated both extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol-3 kinase (PI3-kinase) pathways in SH-SY5Y cells. Ectopic activation of ERK1/2 or PI3-kinase protected against rotenone, whereas inhibition of either pathway attenuated bFGF protection. Reducing the expression of the proapoptotic protein Bcl-2-associated death protein (BAD) by small interfering RNA rendered SH-SY5Y cells resistant to rotenone, implicating BAD in rotenone-induced cell death. Interestingly, bFGF induced a long-lasting phosphorylation of BAD at serine 112, suggesting BAD inactivation through the ERK1/2 signaling pathway. Moreover, primary cultured dopaminergic neurons from mesencephalon were more sensitive to rotenone-induced cell death than nondopaminergic neurons in the same culture. The loss of dopaminergic neurons was blocked by bFGF, an inhibition dependent on ERK1/2 and PI3-kinase signaling. These data suggest that rotenone-induced dopaminergic cell death requires BAD and identify bFGF and its activation of ERK1/2 and PI3-kinase signaling pathways as novel intervention strategies to block cell death in the rotenone model of Parkinson's disease.

Cardiovascular effects of herbicides and formulated adjuvants on isolated rat aorta and heart.

Various formulations of agricultural chemicals, including solutions, wettable powders, and emulsifiable concentrates, contain adjuvants of solvents and surfactants in addition to active ingredients. Among these formulations, herbicides are among the most commonly used pesticides globally. Some pesticides have been demonstrated to cause severe circulatory failure in poisoned humans. To clarify the potential risk of herbicides and their adjuvants influence on the cardiovascular system, four technical grade (TG) herbicides and their end products (EP), including paraquat, glyphosate, glufosinate, and atrazine, as well as their formulated adjuvants isopropylamine (IPA), polyoxyethylene alkylether sulfate (AES), ethyl acetate (EA), xylene, petrolium-170 (P-170), and solvesso-100 (S-100), were assessed to determine their effects on isolated rat aorta and heart. The results revealed that the vasorelaxation effects of the herbicide EPs exceeded those of TGs, and atrazine produced more significant vasorelaxation in rat aortas than the other herbicides tested. The formulated adjuvants of IPA did not affect the aorta; however, AES, EA, xylene, P-170 and S-100 caused significant vasorelaxation. Herbicide EPs-induced vasorelaxation was generally endothelium-dependent. Furthermore, the TG and EP of paraquat, and the TG of glufosinate and glyphosate were found to have no effect on the isolated heart. However, the normal twitch tensions of the isolated heart were significantly inhibited by EPs of glyphosate and glufosinate, and by TG and EP of atrazine. Although, the adjuvants of IPA appeared unaffected, however, AES, EA, xylene, P-170 and S-100 caused complete inhibition and contraction on the isolated hearts. These results indicated that the adjuvants of herbicides might enhance hypotension and contributed to cardiovascular disorders during intoxication.

Effects of sugar cane extract on pseudorabies virus challenge of pigs.

This experiment aimed to evaluate the efficacy of sugar cane extract (SCE) on the modulation of porcine immunity against pseudorabies virus (PrV) infection. Twelve-week-old experimental pigs were fed with SCE (500 mg/kg of body weight per day) for 3 days and challenged with PrV (2 x 10(5) TCID(50)) on the second day. Pigs that were only challenged with PrV and without SCE-treatment served as controls. The leukocyte functional assays were performed on the 7th and 14th day post-PrV challenge. Our results showed a significant enhancement (P<0.05) of natural killer cytotoxicity, lymphocyte proliferation, phagocytic function of monocytes, and interferon-gamma (IFN-gamma) production of CD4(+) and gammadelta T cells in the SCE-treated pigs compared with the controls. In addition, SCE administration reduced the severity of clinical signs and brain lesion in the course of disease in PrV-challenged pigs. SCE-treated pigs showed a 12% growth enhancement compared with untreated controls. SCE administration had an immunostimulating effect on porcine immunity that may subsequently enhance protective activities against PrV infection which may be extensively applied in field for the prevention of infections.

TolC is important for bacterial survival and oxidative stress response in Salmonella enterica serovar Choleraesuis in an acidic environment.

The outer membrane protein TolC, which is one of the key components of several multidrug efflux pumps, is thought to be involved in various independent systems in Enterobacteriaceae. Since the acidic environment of the stomach is an important protection barrier against foodborne pathogen infections in hosts, we evaluated whether TolC played a role in the acid tolerance of Salmonella enterica serovar Choleraesuis. Comparison of the acid tolerance of the tolC mutant and the parental wild-type strain showed that the absence of TolC limits the ability of Salmonella to sustain life under extreme acidic conditions. Additionally, the mutant exhibited morphological changes during growth in an acidic medium, leading to the conflicting results of cell viability measured by spectrophotometry and colony-forming unit counting. Reverse-transcriptional-PCR analysis indicated that acid-related molecules, apparatus, or enzymes and oxidation-induced factors were significantly affected by the acidic environment in the null-tolC mutant. The elongated cellular morphology was restored by adding antioxidants to the culture medium. Furthermore, we found that increased cellular antioxidative activity provides an overlapping protection against acid killing, demonstrating the complexity of the bacterial acid stress response. Our findings reinforce the multifunctional characteristics of TolC in acid tolerance or oxidative stress resistance and support the correlative protection mechanism between oxygen- and acid-mediated stress responses in Salmonella enterica serovar Choleraesuis.

Involvement of NF-κB in regulation of Actinobacillus pleuropneumoniae exotoxin ApxI-induced proinflammatory cytokine production in porcine alveolar macrophages.

Actinobacillus pleuropneumoniae is a crucial respiratory pathogen that causes fibrinous, hemorrhagic, necrotizing pleuropneumonia in pigs. A. pleuropneumoniae exotoxins (ApxI to IV) are the major virulence factors contributing to A. pleuropneumoniae pathogenesis. Previously, we demonstrated that ApxI induces the expression of proinflammatory cytokines in porcine alveolar macrophages (PAMs) via the mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK). Nonetheless, the role of nuclear factor (NF)-κB-a transcription factor widely implicated in immune and inflammatory responses-in ApxI-elicited cytokine production has yet to be defined. In the present study, we examined the involvement of NF-κB in ApxI-elicited production of interleukin (IL)-1ß, IL-8, and tumor necrosis factor (TNF)-α in PAMs and investigated the correlation between NF-κB and MAPK (p38 and JNK) pathways in this event. The results of Western blot analysis, confocal microscopy, and a DNA binding activity assay revealed that the classical NF-κB pathway was activated by ApxI, as evidenced by the decreased levels of IκB and subsequent NF-κB translocation and activation in ApxI-stimulated PAMs. Moreover, the blocking of ApxI-induced NF-κB activation significantly attenuated the levels of mRNA and protein secretion of IL-1ß, IL-8, and TNF-α in PAMs. Notably, the attenuation of JNK activation by a specific inhibitor (SP600125) reduced ApxI-induced NF-κB activation, whereas a p38 blocker (SB203580) had no effect on the NF-κB pathway. Further examination revealed that the level of phosphorylation at serine 536 on the NF-κB p65 subunit was dependent on JNK activity. Collectively, this study, for the first time, demonstrates a pivotal role of NF-κB in ApxI-induced IL-1ß, IL-8, and TNF-α production; JNK, but not p38, may positively affect the activation of the classical NF-κB pathway.

MarA and ramA regulate virulence in Salmonella enterica serovar Choleraesuis.

Salmonella enterica serovar Choleraesuis is considered as an important porcine pathogen that causes serious systemic infections and exhibits poor response to treatment because of an increase in multidrug resistance (MDR). Among the various regulators of resistance, multiple antibiotic resistance factor A (marA) and regulator of acetate metabolism A (ramA) are the most effective in conferring antibiotic tolerance by activation of multidrug efflux pumps. Here we investigated the regulation of virulence in Salmonella Choleraesuis through these two transcriptional regulators. We showed that marA andramA are important for the survival of Salmonella Choleraesuis in an environment of acid and bile salts, since marA- or ramA-deficient Salmonella Choleraesuis strains failed to increase protective responses, as observed by quantitative RT-PCR (qPCR). Further, reduced invasion and survival in host cells was observed in the marA and ramA mutant strains. The results from in vitrostudies with marA- and ramA-deficient strains showed attenuated characteristics in comparison to those in the wild-type strain of Salmonella Choleraesuis when it was used to challenge BALB/c mice. The mutant strains had higher LD50 and presented poor clearance efficiency compared to the parental strain. These findings indicate that MarA and RamA not only regulate drug resistance but also play a role in the virulence of SalmonellaCholeraesuis.