Physiological and molecular responses of antibiotic-resistant Salmonella enterica serovar Typhimurium to acid stress

This study was designed to evaluate the antibiotic susceptibility, lactamase activity, gene expression, and protein profiles of antibiotic-sensitive Salmonella enterica serovar Typhimurium (S. Typhimurium S ) and antibiotic-resistant S. Typhimurium (S. Typhimurium R ) grown at pH 5.5 and 7.3. The antibiotic susceptibility and β-lactamase activity were measured by broth microdilution and nitrocefin assays, respectively. The relative gene expression of efflux pumpand outer membrane-associated genes was measured by real time polymerase chain reaction (RT-PCR), and cell surface proteins were identified by two-dimensional gel electrophoresis (2-DE) analysis. S. Typhimurium R grown at pH 7.3 shows the hyperproduction of β-lactamases regardless of antibiotic resistance. The expression of efflux pumprelated genes (acrA, acrB and tolC) was mostly decreased and increased slightly in antibiotic-treated S. Typhimurium R at pH 7.3. The identified proteins were classified into energy production, amino acid transport and metabolism, posttranslational modification, and carbohydrate transport and metabolism. The results suggest that the antibiotic resistance property of S. Typhimurium under mild acidic condition were highly associated with lactamase activity, efflux pump activity, and cell surface protein profiles. This study provides useful information for better understanding the mechanisms of β-lactam antibiotic-resistant S. Typhimurium exposed to acid stress.


INTRODUCTION
With the increased incidence of bacterial infection, the use of antibiotics has been increased in humans and animals.The extensive use of β-lactam antibiotics has caused the emergence of antibiotic-resistant bacteria.The resistance of Salmonella species to β-lactam antibiotics is caused by i) antibiotic inactivation by βlactamases, ii) decrease in antibiotic permeability, iii) increase in expression of efflux pump-related genes, and iv) alteration of target proteins (Zapun et al., 2008;Sun et al., 2009).More than two mechanisms can commonly be involved in the development of antibiotic resistance in various Gram-negative bacteria (Livermore, 1992;Fernandez-Cuenca et al., 2003).Antibiotic-resistant pathogens in nature can encounter various stress conditions such as acid and additional antibiotics, leading to cross resistance to multiple stresses.The acquired resis-*Corresponding author.E-mail: juheeahn@kangwon.ac.kr.Tel:  tance can alter the physiological and molecular properties in bacteria (Bearson et al., 1998).Therefore, the understanding of phenotypic and genopytic changes is necessary to postulate the fate and impacts of antibioticresistant pathogens under environmental stress conditions.
Salmonella is one of the most serious enteric pathogens, causing severe and life-threatening diseases such as gastroenteritis, septicemia, osteomyelitis, pneumonia, meningitis, and arthritis (Ebong, 1986;Trevejo et al., 2003;Brent et al., 2006).The enteric pathogens can survive through acidic stomach conditions and reach the intestinal tract, which plays an important role in pathogenesis (Bearson et al., 1998).
The low pH enables S. Typhimurium to induce an acid tolerance response (Foster and Spector, 1995;Greenacre et al., 2006).However, there have been relatively few studies on the stress responses of antibiotic resistant S. Typhimurium exposed to mild acidic conditions.Therefore, the objec-tive of this study was to characterize the physiological properties of antibioticresistant Salmonella grown at the mild acidic stress as assessed by the role of β-lactamase production, efflux pump-and outer membrane-associated genes, and cell surface proteins with were compared with antibioticsensitive S. Typhimurium.

Bacterial strains and culture conditions
Strains of Salmonella enterica serovar Typhimurium KCCM 40253 and Salmonella Typhimurium CCARM 8009 were purchased from the Korean Culture Center of Microorganisms (KCCM, Seoul, Korea) and the Culture Collection of Antimicrobial Resistant Microbes (CCARM, Seoul, Korea), respectively.The strains were cultivated in trypticase soy broth [Tryptic Soy Broth (TSB); Becton Dickinson, Sparks, MD, USA] at 37°C for 20 h.After cultivation, cultures were diluted with TSB (pH 7.3) and TSB adjusted to pH 5.5 by lactic acid (Shinyo Pure Chemical, Osaka, Japan).

Antibiotic susceptibility testing
Six β-lactam antibiotics, including ampicillin, cefoxitin, ceftazidime, cephalothin, penicillin G, and piperacillin, were purchased from the Sigma Chemical Co.(St.Louis, MO, USA).All antibiotic stock solutions were prepared at the final concentrations of 256 µg/ml in TSB (pH 7.3) and TSB adjusted to pH 5.5 by lactic acid.The antibiotic susceptibility of the strains grown at pH 5.5 and 7.3 was expressed by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).The MICs of β-lactam antibiotics were determined by the broth microdilution method.The β-lactam antibiotics were serially diluted (1:2) from 256 to 0.25 µg/ml with TSB (pH 7.3) in 96-well plates.MIC and MBC values were determined as the lowest concentrations of antibiotic with no visible growth after incubation and with negative results in enrichment tests, respectively.

Hydrolysing activity of β-lactamases
The β-lactamase activity was evaluated by nicrocefin hydrolysis Cho and Ahn 579 assay (Arora and Bal, 2005).Bacterial cells (10 µl) exposed to a half MIC were incubated with 25 µl of 1.5 mM nitrocefin in 96 well plate for 3 h at 37°C.After incubation, the cells were diluted with 100 µl of 50 mM phosphate buffer (pH 7.4) to measure the absorbance at 515 nm using a microplate reader (BioTek Instruments, Inc.,Winooski, VT, USA).

Quantitative real time -polymerase chain reaction (RT-PCR)
S. Typhimurium S and S. Typhimurium R were grown in TSB (pH 5.5 and 7.3) containing a half MIC of each antibiotic (ampicillin, cefoxitin, ceftazidime, cephalothin, penicillin G, and piperacillin) at 37°C for 20 h.The bacterial cultures (10 7 CFU/ml, 500 µl) were mixed with 1 ml of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) and centrifuged at 5,000 g for 10 min (He and Ahn, 2011).RNA was extracted according to RNeasy ® Mini Handbook (Qiagen).The RNA sample (12 µl) was mixed with genomic DNA Wipeout buffer at 42°C for 2 min and mixed with a master mixture containing 1 µl of Quantiscript reverse transcriptase, 4 µl of Quantiscript RT buffer, and 1 µl of RT primer mix.The reaction mixture was incubated at 42°C for 15 min and then incubated at 95°C for 3 min to inactivate the reverse transcriptase.The oligonucleotide primers shown in Table 1 were synthesized by IDT (Integrated DNA Technologies, Coralville, IA, USA) for this study.The reaction mixture (20 μl) containing of 2× QuantiTect SYBR Green PCR Master (10 μl), 60 pmol primer (0.6 μl), cDNA (2 μl), and RNase-free water (6.8 µl) was amplified using an iCycler iQ™ system (Bio-Rad Laboratories, Hemel Hempstead, UK).The reaction mixture was denatured initially (95°C, 15 min), followed by 45 thermal cycles of (94°C, 15 s; 59°C, 20 s; and 72°C, 15 s).The melt-curve analysis was performed immediately after amplification protocol with 0.4°C increments per 10 s for 85 cycles from 65 to 97°C.The PCR products were visualized and analyzed using the iQ5 PCR detection system (Bio-Rad Laboratories), then calculated by 2 -ΔΔC T method (Livak and Schmittgen, 2001).

Two-dimensional gel electrophoresis (2-DE) analysis
Bacterial cells (300 µl each) grown at pH 5.5 and 7.3 were collected by centrifugation at 5,000 g for 10 min and resuspended in lysis buffer containing protease inhibitors.The lysates were homogenized by sonication for 10 min on ice, incubated for 1 h at 15°C, and ultra-centrifuged at 20,000 g for 1 h.The collected proteins (500 µg) were loaded onto immobilized pH gradient (IPG) strips (pH 3 to 10 nonlinear; Amersham Pharmacia Biotech, Uppsala, Sweden) and isoelectric focusing (IEF) was carried out at 20°C for 80,000 Vh.After IEF, the second dimension was resolved on 9 to 17% linear gradient polyacrylamide gels and stained with coomassie blue G250 (Bio-Rad; Hercules, CA, USA) for 24 h.Gels were scanned in Bio-Rad G710 densitometer (Richmond, CA, USA) and converted into 12-bit TIFF file.The images were analyzed with ImageMaster TM 2D Platinum software (Amersham Pharmacia Biotech).The gel patterns were compared to the reference gel based on the spot intensities.The normalized spots with more than 2-fold variation were defined as differentially expressed proteins.

Protein identification
Proteins were identified using a mass fingerprinting.The spots were excised from gels, destained, and digested with trypsin (Promega, Madison, WI, USA).The peptide mass fingerprinting (PMF) was analyzed using a Voyager DE-PRO MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA).Monoisotopic peptide masses were obtained using Data Explorer 4.4 (PerSeptive Biosystems; Foster City, CA, USA).Proteins were identified using a

Statistical analysis
All experiments were performed in three replicates.Data were analyzed using the general linear model (GLM) and the least significant difference (LSD) procedures of the Statistical Analysis System (SAS).Significant mean differences among treatments were compared by Fisher's LSD at p<0.05.

Acid-induced antibiotic susceptibility and β-lactam hydrolyzing activity
The antibiotic susceptibility of S. Typhimurium S and S. Typhimurium R grown at pH 7.3 and 5.5 was evaluated against different β-lactam antibiotics (Table 2).Compared with S. Typhimurium S , S. Typhimurium R was highly resistant to ampicillin, cefoxitin, cephalothin, penicillin G, and piperacillin at pH 5.5 and 7.3.S. Typhimurium S at pH 5.5 was more resistant to ceftazidime than S. Typhimurium R .S. Typhimurium S and S. Typhimurium R were highly sensitive to most antibiotics at pH 7.3.This might be attributed to the fact that the optimum temperature and pH can enhance the antibiotic activity resulting in the increased susceptibility of S. Typhimurium S to antibiotics (Russell, 2003).S. Typhimurium R had the highest resistance to ampicillin (pH 5.5 and 7.3) and penicillin G (pH 5.5), showing more than 256 μg/ml of MICs.The antibiotic-resistant S. Typhimurium R grown at pH 5.5 was more resistant to cefoxitin, ceftazidime, and cephalothin than that grown at pH 7.3.This implies that the antibiotic-resistant strains can acquire adaptation to acid stress, leading to the induc- a -, OD < 0.3; +, 0.3≤ OD <0.5; ++, 0.5 ≤ OD < 1.0; +++, 1.0 ≤ OD < 1.5; ++++, 1.5 ≤ OD.
tion of cross-protection phenomenon (Leyer and Johnson, 1993;Greenacre and Brocklehurst, 2006).The antibiotic resistance can be increased with lowering pH levels due to the changes in cell surface hydrophobicity (Laub et al., 1989).The enzymatic inactivation of antibiotics is one possible mechanism of acquired antibiotic resistance in bacteria (Tenover, 2006).The differences in resistance between two strains were noticeable to ampicillin (seven to eight fold), penicillin G (four to five fold), and piperacillin (four to six fold) at pH 5.5 and 7.3.The β-lactam hydrolyzing activities of β-lactamases were evaluated in S. Typhimurium S and S. Typhimurium R grown at pH 5.5 and 7.3 as shown in Table 3.The hydrolyzing activities were stronger in S. Typhimurium R grown at pH 5.5 and 7.3 than S. Typhimurium S .The hyperproduction of β-lactamases was observed in S. Typhimurium R at pH 7.3 (Table 3).S. Typhimurium R grown at both pH 5.5 and 7.3 shows the highest hydrolyzing activity against cephalothin.The considerable increase in β-lactamase activity was observed in S. Typhimurium R grown at pH 7.3.The acidic pH condition causes the increase in ionization constant responsible for the loss of lactamase activity (Ohsuka et al., 1995).The lactamase activity corresponded with the antibiotic resistance of S. Typhimurium R , suggesting that the βlactam resistance can be most possibly involved in the enzymatic inactivation mechanism (Li and Nikaido, 2009).The observation that piperacillin had a relatively low lactamase activity suggests the resistance of S. Typhimurium R to piperacillin might be produced by other mechanisms (Shannon and Phillips, 1986, Phillips and Shannon, 1993, Stapleton et al., 1995).Cefoxitin and cephalothin are known as strong β-lactamase inducers in Gram-negative bacteria, while piperacillin is a poor inducer (Shannon and Phillips, 1986).
Typhimurium S and S. Typhimurium R grown at the half MIC of each β-lactam antibiotic at pH 5.5 and 7.3 (Figure 1).The relative expression levels of acrA and tolC genes were changed by less than 1-fold in S. Typhimurium S and S. Typhimurium R exposed to most β-lactam antibiotic at pH 5.5 and 7.3 with the exception of S. Typhimurium R exposed to piperacillin at pH 5.5, showing 1.86-fold decrease in acrA and 2.75-fold decrease in tolC (Figure 1f).The AcrAB-TolC tripartite complex, belonging to the resistance-nodulation-cell division (RND) family, plays an important role in antibiotic resistance and bacterial virulence through antibiotic extrusion (Baucheron et al., 2004;Nikaido et al., 2008).The acrB gene was significantly down-regulated in S. Typhimurium R exposed to ceftazidime (1.77-fold) and piperacillin (2.75-fold) at pH 5.5 (Figuire 1c and 1f).
This result implies that the AcrAB-TolC efflux pump system was not directly involved in the β-lactam resistance of S. Typhimurium R at pH 5.5 and 7.3.The relative expression levels of ompD were increased by more than 1-fold in the ampicillin-treated S. Typhimurium S and S. Typhimurium R at both pH 5.5 and 7.3 (Figure 1a).The ompD gene was highly up-regulated in S. Typhimurium R exposed to cephalothin (2.79-fold) and piperacillin (3.52-fold) at pH 7.3 (Figure 1d and f), leading to the increased in outer membrane permeability (Santiviago et al., 2003).The hilA gene was significantly down-regulated in the ampicillin-treated Typhimurium S at pH 5.5 (1.30-fold), the cefoxitin-treated Typhimurium S at pH 7.3 (1.04-fold), the cephalothin-treated S. Typhimurium R at pH 5.5 (1.21-fold), the piperacillintreated S. Typhimurium S at pH 5.5 (1.12-fold), and the piperacillin-treated S. Typhimurium R at pH 5.5 (2.00-fold) (Figure 1a, b, d, and f).The hilA gene encoding Salmonella Pathogenicity Island (SPI) was up-regulated in S. Typhimurium R exposed to ampicillin, ceftazidime, cephalothin, penicillin, and piperacillin at pH 7.3.HilA can regulate the Type III secretion system (TTSS), which contributes to the bacterial invasion of epithelial cells (Durant et al., 2000, Phoebe Lostroh andLee, 2001;Boddicker et al., 2003).The significant increase in the fliA gene expression was observed in the cefoxitin-treated S. Typhimurium R at pH 5.5 (3.37-fold) (Figure 1b).The   expression of fimA gene is associated with the adherence and invasion abilities of bacteria, which is transcriptionally regulated by environmental stresses (Xie et al., 1997).

R under acid stress
The 2-DE profiles were used to evaluate the changes in protein expression in antibiotic-treated S. Typhimurium S and S. Typhimurium R at pH 5.5 and 7.3 (Figure 2).The numbers of spots in S. Typhimurium S at pH 5.5, S. Typhimurium S at pH 7.3, S. Typhimurium R at pH 5.5, and S. Typhimurium R at 7.3 were 413, 336, 216, and 212, respectively, which were distributed in the pH ranges of 3 to 10 and molecular weight from 10 to 200 kDa.When compared with S. Typhimurium S at pH 7.3 (reference strain), 251 spots were paired with S. Typhimurium S at pH were identified as shown in Table 4.The identified proteins were categorized based on their functional properties, including energy production and conversion (adhE, pflB, and ordL), amino acid transport and metabolism (cadA and asd), posttranslational modifycation (clpB and surA), cell motility (fliC and flgL), and carbohydrate transport and metabolism (pgk, tpiA, and ptsH) (Table 4).The observation that the expression of lysine decarboxylase was increased in S. Typhimurium S and S. Typhimurium R at pH 5.5 confirms previous report that the cadA gene was overexpressed in S.
Typhimurium exposed to acid stress (Greenacre et al., 2006).The increased activity of lysine decarboxylase may result in the enhanced production of cadaverine and suppression of permeability, which blocks the penetration of antibiotics into the bacterial cells (Tkachenko et al., 2009).The inactivation of periplasmic prolyl isomerase (SurA) affects an enhanced antibiotic susceptibility (Justice et al., 2005).The distinctive proteins (SipA, SipB, TraI, FliC, Frr, Int, and SolA) expressed in S. Typhimurium R might be related to the enhanced antibiotic resistance.

Conclusion
This study highlights the potential impact of acid and antibiotic stress conditions on the changes in the antibiotic susceptibility, gene expression, and protein profile.The mechanisms of antibiotic resistance in bacteria are complex and associated with many factors including environmental conditions and bacterial states.
The production of β-lactamases and the enhanced expression of efflux-related genes at the transcriptional and translational levels are certainly related to the antibiotic resistance and virulence.However, the results obtained from the limited set of S. Typhimurium S and S. Typhimurium R strains are not enough to conclude their exact mechanisms associated with the antibiotic susceptibilities, gene expression levels, and cellular surface proteins in response to acid and antibiotic stresses.Nevertheless, this study sheds new light on our understanding of diverse physiogenetic characteristics of antibiotic-resistant pathogens under different environ-mental stress conditions.

Figure 2 .
Figure 2. Two-DE images of cell surface proteins extracted from S. Typhimurium S at pH 5.5 (a), S. Typhimurium S at pH 7.3 (b), S. Typhimurium R at pH 5.5 (c), and S. Typhimurium R at pH 7.3 (d).Representative acid-induced and acid-repressed proteins are assigned in the spot numbers, 1-18 and 19-31, respectively.Non-paired proteins are indicated by arrows with letters.

Table 1 .
Primers used in real-time RT-PCR analysis for S. Typhimurium a F, forward; R, reverse.

Table 3 .
Hydrolysing activity a of the lactamase produced from S. Typhimurium strains exposed to a half MIC of selected antibiotics

Table 4 .
Protein identification of paired and non-paired spots in 2 DE of S. Typhimurium strains exposed to acid stress.