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CA Lenz, CM Hew Ferstl, RF Vogel (2010)
Sub-lethal stress effects on virulence gene expression in Enterococcus faecalisFood Microbiol, 27
VK Juneja, HP Dwivedi, X Yan (2012)
Novel natural food antimicrobialsAnnu Rev Food Sci Technol, 3
TF Mah, GA O’Toole (2001)
Mechanisms of biofilm resistance to antimicrobial agentsTrend Microbiol, 9
ME Dominiecki, J Weiss (1999)
Antibacterial action of extracellular mammalian group IIA phospholipase A2 against grossly clumped Staphylococcus aureusInfect Immun, 67
MN Bellon-Fontaine, J Rault, CJ Oss (1996)
Microbial adhesion to solvents: a novel method to determine the electron-donor/electron-acceptor or Lewis acid–base properties of microbial cellsColloids Surf B, 7
N Merino, A Toledo-Arana, M Vergara-Irigaray, J Valle, C Solano, E Calvo, JA Lopez, TJ Foster, JR Penades, I Lasa (2009)
Protein A-mediated multicellular behavior in Staphylococcus aureusJ Bacteriol, 191
R Houdt, C Michiels (2010)
Biofilm formation and the food industry, a focus on the bacterial outer surfaceJ Appl Microbiol, 109
JW Costerton, Z Lewandowski, DE Caldwell, DR Korber, HM Lappin-Scott (1995)
Microbial biofilmsAnnu Rev Microbiol, 49
SB Hernandez, I Cota, A Ducret, L Aussel, J Casadesus (2012)
Adaptation and preadaptation of Salmonella enterica to bilePLoS Genet, 8
Z Saldana, J Xicohtencatl-Cortes, F Avelino, AD Phillips, JB Kaper, JL Puente, JA Giron (2009)
Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curliEnviron Microbiol, 11
MG Lei, D Cue, CM Roux, PM Dunman, CY Lee (2011)
Rsp inhibits attachment and biofilm formation by repressing fnbA in Staphylococcus aureus MW2J Bacteriol, 193
KJ Livak, TD Schmittgen (2001)
Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT methodMethods, 25
DP O’Connell, T Nanavaty, D McDevitt, S Gurusiddappa, M Hook, TJ Foster (1998)
The fibrinogen-binding MSCRAMM (clumping factor) of Staphylococcus aureus has a Ca2+-dependent inhibitory siteJ Biol Chem, 273
S Bhakdi, J Tranum-Jensen (1991)
Alpha-toxin of Staphylococcus aureusMicrobiol Rev, 55
WM Dunne, EO Mason, SL Kaplan (1993)
Diffusion of rifampin and vancomycin through a Staphylococcus epidermidis biofilmAntimicrob Agents Chemother, 37
AY Rad, H Ayhan, E Piskin (1998)
Adhesion of different bacterial strains to low-temperature plasma treated biomedical PVC catheter surfacesJ Biomater Sci, 9
FG Sorroche, MB Spesia, A Zorreguieta, W Giordano (2012)
A positive correlation between bacterial autoaggregation and biofilm formation in native Sinorhizobium meliloti isolates from ArgentinaAppl Environ Microbiol, 78
B Re, B Sgorbati, M Miglioli, D Palenzona (2000)
Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium logumLett Appl Microbiol, 31
J Huang, PW O’Toole, W Shen, H Amrine-Madsen, X Jiang, N Lobo, LM Palmer, L Voelker, F Fan, MN Gwynn, D McDevitt (2004)
Novel chromosomally encoded multidrug efflux transporter MdeA in Staphylococcus aureusAntimicrob Agents Chemother, 48
JB Kaplan (2011)
Antibiotic-induced biofilm formationInt J Artif Organs, 34
Ann Microbiol (2013) 63:1213–1217 DOI 10.1007/s13213-012-0572-y SHORT COMMUNICATION Physiochemical and molecular properties of antimicrobial-exposed Staphylococcus aureus during the planktonic-to-biofilm transition Hyeon-Yong Lee & Yunyun Zou & Juhee Ahn Received: 2 August 2012 /Accepted: 12 November 2012 /Published online: 25 November 2012 Springer-Verlag Berlin Heidelberg and the University of Milan 2012 Abstract This study was designed to characterize the phys- Surface-associated bacterial communities, known as micro- icochemical and molecular properties of Staphylococcus bial biofilms, are present ubiquitously in natural ecosystems. aureus cells treated with nisin, allyl isothiocyanate Biofilms are embedded in a self-secreted matrix consisting (AITC), thymol, eugenol, and polyphenol during the transi- of extracellular polymeric substances comprised of proteins, tion from planktonic to biofilm growth as measured by nucleic acids, and polysaccharides (Costerton et al. 1995). hydrophobicity, auto-aggregation, and differential gene ex- The development of complex and dynamic biofilm struc- pression. Thymol exhibited the highest antimicrobial activ- tures involves mainly (1) attachment, (2) maturation, and (3) ity against planktonic, biofilm-forming, biofilm, and dispersion, which can be influenced by cellular properties dispersed cells, showing 0.21, 0.22, 0.46, and 0.26 mg/ml (hydrophobicity, aggregation, and slime production) and of MIC values, respectively. The lowest hydrophobicity was environmental stresses (low pH, starvation, and preserva- observed in planktonic cells treated with polyphenol (16 %), tives) (Costerton et al. 1995; Saldana et al. 2009). Since followed by thymol (29 %). The auto-aggregation abilities biofilm cells are highly resistant to stress conditions and were more than 85 % for nisin, AITC, eugenol, polyphenol, thus responsible for persistent and chronic bacterial infection, and the control. The cell-to-surface interaction was related biofilm-associated infections have received great attention in positively to biofilm formation by S. aureus. The adhesion- the field of medicine and in food processing industries related gene (clfA), virulence-related genes (spa and hla), (Van Houdt and Michiels 2010). and efflux-related gene (mdeA) were down-regulated in both Antimicrobial preservatives have long been used to ex- planktonic and biofilm cells treated with AITC, thymol, and tend the shelf-life and improve the safety and quality of food eugenol. The results suggest that the antimicrobial tolerance products. Recently, the use of naturally occurring preserva- and virulence potential were varied in the cell states during tives has become more attractive to the food industry due to the planktonic-to-biofilm transition. This study provides use- consumer demand for safe foods. Many natural substances, ful information for understanding the cellular and molecular such as nisin, allyl isothiocyanate (AITC), thymol, eugenol, responses of planktonic and biofilm cells to antimicrobial- and polyphenol, are well recognized to possess broad- induced stress. spectrum antimicrobial, anti-biofilm, antioxidant, anti- inflammatory, and antitumor activities when used alone or . . . Keywords Staphylococcus aureus Planktonic Biofilm in combination (Juneja et al. 2012). The prolonged use of . . Hydrophobicity Auto-aggregation Gene expression antimicrobials can alter the physiological molecular proper- ties of bacterial cells, resulting in enhanced tolerance and adaptation to stresses (Lenz et al. 2010; Hernandez et al. H.-Y. Lee 2012). Nevertheless, there have been relatively few studies Department of Teaics, Seowon University, Chungju 361-742, on the cellular and molecular responses of foodborne patho- Republic of Korea gens to antimicrobial-induced stresses during the planktonic– Y. Zou J. Ahn (*) biofilm transition. Therefore, the aim of this study was to Department of Medical Biomaterials Engineering, evaluate the physiological and molecular properties of Kangwon National University, Chuncheon, Gangwon 200-701, antimicrobial-treated Staphylococcus aureus during the Republic of Korea transition from planktonic to biofilm-detached cells. e-mail: juheeahn@kangwon.ac.kr 1214 Ann Microbiol (2013) 63:1213–1217 Antimicrobials, including nisin produced by Lactococcus (PBS, pH 7.2) at 0.5 OD (A ) and incubated at 600 0h lactis, AITC (95 %), thymol (99.5 %), eugenol (98 %), and 37 °C for 2 h. After incubation, the absorbance of the super- polyphenol extracted from green tea, were purchased from natant was measured at 600 nm (A ) using a microplate 2h Sigma-Aldrich (St. Louis, MO). Staphylococcus aureus reader (Molecular Devices, Menlo Park, CA). The auto- strain KACC 10196 was obtained from the Korean Agricul- aggregation was estimated by the following formula: tural Culture Collection (KACC; Suwon, Korea) and culti- AutoaggregationðÞ % ¼ðÞ 1 A =A 100: 2h 0h vated in trypticase soy broth supplemented with 0.1 % yeast The hydrophobicity of planktonic cells was determined extract (TSBY, Difco, Detroit, MI) at 37 °C for 20 h. The according to the bacterial affinity to solvent assay (Bellon- cultures were collected by centrifugation at 10,000 g for Fontaine et al. 1996). The planktonic cells (2 ml) cultured at 10 min at 4 °C and used as a planktonic indicator strain 37 °C for 24 h in the absence or presence of each antimi- for auto-aggregation, hydrophobicity, and antimicrobial sus- crobial at one-half the MIC were resuspended in PBS ceptibility tests. In order to obtain biofilm cells (10 CFU/ml), (pH 7.2) to 0.5 OD (A ). The suspensions were mixed 600 0min S. aureus planktonic cells were cultured in TSB on 24-well with hexadecane (1 ml) and allowed to stand for 20 min flat-bottomed polystyrene microtiter plates (BD Falcon, San to separate the aqueous phase. The aqueous phase was Jose, CA). measured at 600 nm (A ) using a microplate reader 20min The susceptibility of S. aureus planktonic and biofilm (Molecular Devices). The hydrophobicity was estimat- cells to each antimicrobial was evaluated using a broth ed by the following formula: HydrophobicityðÞ % ¼ dilution method (CLSI 2009). The planktonic or biofilmðÞ 1 A =A 100: 20 min 0min cells were cultured at 37 °C for 24 h in TSB containing 0 The planktonic or biofilm cells (0.5 ml) cultured at one- to 5.15 kIU/ml (or mg/ml) of nisin, AITC, thymol, eugenol, half the MICs of nisin, AITC, thymol, eugenol, and poly- or polyphenol in 24-well plates. After 24-h incubation, phenol were mixed with 1 ml RNAprotect Bacteria Reagent planktonic, biofilm, and dispersed cells were collected sep- (Qiagen, Hilden, Germany), centrifuged at 5,000 g for arately by centrifugation at 5,000 g for 10 min at 4 °C or 10 min, and mixed with a buffer containing guanidine iso- scraping using a cell scraper (SPL Life Science, Pochen, thiocyanate, and then lysed with a buffer containing lyso- Korea). The collected cells were serially (1:10) diluted with zyme. The lysates were mixed with ethanol and then loaded 0.1 % sterile buffered peptone water (BPW), plated on the to an RNeasy mini column to extract RNA. The cDNA was trypticase soy agar (TSA; Difco) using an Autoplate® Spiral synthesized according to the QuantiTech Reverse Transcrip- Plating System (Spiral Biotech, Norwood, MA), and incu- tion procedure (Qiagen). The RNA extract was mixed with a bated at 37 °C for 24–48 h to enumerate viable planktonic, master mixture of reverse transcriptase, RT buffer, and RT biofilm, and detached cells using a QCount® Colony Counter primer mix and then incubated at 42 °C for 15 min, followed (Spiral Biotech). The minimum inhibitory concentration by 95 °C for 3 min. The PCR mixture (20 μl) containing (MIC) was defined as the lowest concentration (kIU/ml or 10 μl of 2× QuantiTect SYBR Green PCR Master, 60 pmol mg/ml) of each antimicrobial at which the growth was primer (0.6 μl), cDNA (2 μl), and RNase-free water (6.8 μl) inhibited by 99 % compared to the control. was amplified by an iCycler iQ™ system (Bio-Rad Labora- The auto-aggregation assay was used to determine cell- tories, Hemel Hempstead, UK) and then denatured at 95 °C to-cell surface interaction (Del Re et al. 2000). Planktonic for 15 min, followed by 45 cycles of 94 °C for 15 s, 59 °C cells were cultured at 37 °C for 24 h in the absence or for 20 s, and 72 °C for 15 s. The PCR products were presence of each antimicrobial at one-half the MIC. The analyzed using the iQ5 real-time PCR detection system cultures were suspended in phosphate-buffered saline (Bio-Rad). The relative expression of targeted genes was Table 1 Minimum inhibitory concentrations (MIC , mg/ml) of se- dispersed cells. Means with different letters within a row (x–z) and a lected antimicrobials against different states of Staphylococcus aureus column (a–d) are significantly different at P<0.05 during the transitions from planktonic, biofilm-forming, biofilm to Treatment Staphylococcus aureus Planktonic cells Biofilm-forming cells Biofilm cells Biofilm-detached cells Nisin 0.35±0.10z bc 0.26±0.04z c 1.94±0.05x b 0.84±0.24y c AITC 0.87±0.15y b 0.95±0.05y b 3.84±0.69x a 3.54±0.33x a Thymol 0.21±0.03y c 0.22±0.02y c 0.46±0.06x c 0.26±0.04y c Eugenol 0.40±0.04y bc 0.37±0.09y c 1.66±0.31x b 0.62±0.13y c Polyphenol 1.50±0.45y a 2.26±0.37y a 4.18±0.54x a 2.62±0.56y b The unit for the nisin is kIU/ml Ann Microbiol (2013) 63:1213–1217 1215 estimated by the comparative C method (Livak and System (SAS) software (SAS Institute, Cary, NC). Significant Schmittgen 2001). The ΔC values of the target genes were mean differences among treatments were determined by estimated by normalizing to the endogenous reference gene Fisher’s least significant difference (LSD) at P<0.05. (16S rRNA). The antimicrobial susceptibility of S. aureus planktonic, All analyses were performed in duplicate on three repli- biofilm-forming, biofilm-formed, and dispersed cells was cates. Data were analyzed using the Statistical Analysis evaluated based on the MIC values of antimicrobials as ab 8 8 6 6 4 * 4 AB BC 2 2 0 0 A m m A A mn -2 -2 -4 -4 -6 -6 -8 -8 clfA spa hla mdeA clfA spa hla mdeA c d 8 8 6 6 4 4 2 2 0 0 m m -2 -2 A A AB mn n B -4 -4 -6 -6 -8 -8 clfA spa hla mdeA clfA spa hla mdeA * * AB A -2 -4 -6 no -8 clfA spa hla mdeA Fig. 1 Relative gene expression in Staphylococcus aureus planktonic letters within planktonic (A–C; open bars) and biofilm (m–o; shaded cells (open bars) and biofilm cells (shaded bars) grown at a half bars) cells on the bars are significantly different at P<0.05. * Signif- minimum inhibitory concentrations (MIC) of nisin (a), AITC (b), icant difference in gene expression levels between planktonic and thymol (c), eugenol (d), and polyphenol (e). Means with different biofilm cells at P<0.05 (n010) Fold change in gene expression Fold change in gene expression Fold change in gene expression Fold change in gene expression Fold change in gene expression 1216 Ann Microbiol (2013) 63:1213–1217 shown in Table 1. Staphylococcus aureus biofilm cells were planktonic and biofilm cells treated with polyphenol highly resistant to nisin, AITC, thymol, eugenol, and poly- (Fig. 1e). The attachment ability of bacterial cells to biotic phenol, showing 1.94, 3.84, 0.46, 1.66, and 4.18 mg/ml, or abiotic surface is regulated by biofilm-associated proteins respectively, followed by biofilm-detached, biofilm- such as FnbA, Cna, and ClfA (Lei et al. 2011). ClfA can forming, and planktonic cells. The lowest MIC values were promote the attachment of fibrinogen or fibrin to bacterial observed for thymol against planktonic cells (0.21 mg/ml), cells, leading to enhanced virulence and phagocytosis resis- biofilm-forming cells (0.22 mg/ml), biofilm cells (0.46 mg/ tance (O’Connell et al. 1998; Dominiecki and Weiss 1999). ml), and biofilm-detached cells (0.26 mg/ml). The antimi- The archetypal cell-wall-anchored protein (Spa) plays a crobial susceptibility varied with the states of bacterial cells. significant role in biofilm formation, and is involved with This observation is in good agreement with the general the induction of bacterial aggregation (Merino et al. 2009). characteristic of biofilm cells, which are highly resistant to The cytotoxic alpha-hemolysin (Hla) is involved in invasive- environmental stresses (Mah and O’Toole 2001;Kaplan ness and virulence of S. aureus (Bhakdi and Tranum-Jensen 2011). The enhanced resistance of S. aureus biofilm cells 1991). Enhanced antimicrobial resistance is associated with to antimicrobials might be due to their structural properties, the overexpression of mdeA gene, a multidrug efflux pump resulting in inefficient diffusion of antimicrobials into the from S. aureus (Huang et al. 2004). cells (Dunne et al. 1993). In conclusion, this study underlines the physicochemical The physicochemical properties of S. aureus planktonic and molecular properties of planktonic biofilm-forming S. cells grown at the MIC of each antimicrobial was evaluated aureus, the biofilm, and detached cells and their association by cell surface hydrophobicity and auto-aggregation. The with antimicrobial tolerance and virulence potential. The planktonic cells treated with antimicrobials showed lower main findings of this study were that (1) biofilm cells and hydrophobicity than the control (85 %). Hydrophobicity biofilm-dispersed cells were highly resistant to nisin, AITC, was reduced significantly in cells treated with polyphenol thymol, eugenol, and polyphenol; (2) the cell-to-surface (16 %), followed by thymol (29 %), nisin (59 %), AITC interaction (hydrophobicity) varied with antimicrobial treat- (59 %), and eugenol (71 %). The highest biofilm-forming ments and was associated positively with biofilm-forming ability was observed in S. aureus cells treated with polyphe- ability; (3) thymol effectively inhibited S. aureus cells at nol, followed by thymol. The highly hydrophilic property of various states; (4) the increased antimicrobial resistance and S. aureus planktonic cells treated with polyphenol may virulence potential were attributed to the up-regulation of contribute to the irreversible adhesion to the hydrophobic biofilm-related gene (clfA), virulence-related genes (spa and polystyrene surface. This confirms that the hydrophilic cell hla), and efflux-related gene (mdeA). The results of this surface can be correlated positively with biofilm-forming study shed new light on our understanding of different states ability (Rad et al. 1998). Compared to the control (92 %), of foodborne pathogens exposed to stresses in terms of the auto-aggregation ability was not significantly different physicochemical and molecular properties. from that of cells treated with nisin (95 %), AITC (92 %), eugenol (87 %), and polyphenol (88 %). The auto-aggregation Acknowledgment This study was supported by the Korea Institute ability of bacterial cells correlated positively with biofilm- of Planning & Evaluation for Technology (IPET) in Food Agriculture, forming ability (Sorroche et al. 2012). However, there was Forestry & Fisheries (Grant No. 110130-03-2-HD110). no noticeable relationship between auto-aggregation and biofilm-forming ability of antimicrobial-treated S. aureus planktonic cells in this study, which suggests that antimicro- bials can inhibit biofilm formation at different target sites. References The relative expression of adhesion-related gene (clfA), virulence-related genes (spa and hla), and efflux-related Bellon-Fontaine MN, Rault J, van Oss CJ (1996) Microbial adhesion to gene (mdeA) were observed in planktonic and biofilm cells solvents: a novel method to determine the electron-donor/electron- of S. aureus treated with antimicrobials (Fig. 1). As shown acceptor or Lewis acid–base properties of microbial cells. Colloids in Fig. 1a, the relative expression levels of clfA, spa, hla, Surf B 7:47–53 Bhakdi S, Tranum-Jensen J (1991) Alpha-toxin of Staphylococcus and mdeA genes increased to 2.3, 0.9, 1.5, and 1.9-fold in aureus. Microbiol Rev 55:733–751 nisin-treated S. aureus planktonic cells, while they were CLSI (2009) Methods for dilution antimicrobial susceptibility tests for down-regulated in biofilm cells. The genes, clfA, spa, hla, bacteria that grow aerobically. Approved standard M07-A8. and mdeA, were down-regulated in AITC-, thymol-, and Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin- Scott HM (1995) Microbial biofilms. Annu Rev Microbiol eugenol-treated S. aureus planktonic and biofilm cells as 49:711–745 shown in Fig. 1b–d.The clfA, spa,and hla genes were Del Re B, Sgorbati B, Miglioli M, Palenzona D (2000) Adhesion, down-regulated in the biofilm cells treated with polyphenol autoaggregation and hydrophobicity of 13 strains of Bifidobacte- (>5-fold), while the mdeA gene was overexpressed in both rium logum. Lett Appl Microbiol 31:438–442 Ann Microbiol (2013) 63:1213–1217 1217 Dominiecki ME, Weiss J (1999) Antibacterial action of extracellular Mah TF, O’Toole GA (2001) Mechanisms of biofilm resistance to mammalian group IIA phospholipase A2 against grossly clumped antimicrobial agents. Trend Microbiol 9:34–39 Staphylococcus aureus. Infect Immun 67:2299–2305 Merino N, Toledo-Arana A, Vergara-Irigaray M, Valle J, Solano C, Dunne WM, Mason EO, Kaplan SL (1993) Diffusion of rifampin and Calvo E, Lopez JA, Foster TJ, Penades JR, Lasa I (2009) Protein vancomycin through a Staphylococcus epidermidis biofilm. Anti- A-mediated multicellular behavior in Staphylococcus aureus.J microb Agents Chemother 37:2522–2526 Bacteriol 191:832–843 Hernandez SB, Cota I, Ducret A, Aussel L, Casadesus J (2012) O’Connell DP, Nanavaty T, McDevitt D, Gurusiddappa S, Hook M, Adaptation and preadaptation of Salmonella enterica to bile. Foster TJ (1998) The fibrinogen-binding MSCRAMM (clumping 2+ PLoS Genet 8:1–15 factor) of Staphylococcus aureus has a Ca -dependent inhibitory Huang J, O’Toole PW, Shen W, Amrine-Madsen H, Jiang X, Lobo N, site. J Biol Chem 273:6821–6829 Palmer LM, Voelker L, Fan F, Gwynn MN, McDevitt D (2004) Novel Rad AY, Ayhan H, Piskin E (1998) Adhesion of different bacterial chromosomally encoded multidrug efflux transporter MdeA in Staph- strains to low-temperature plasma treated biomedical PVC cathe- ylococcus aureus. Antimicrob Agents Chemother 48:909–917 ter surfaces. J Biomater Sci 9:915–929 Juneja VK, Dwivedi HP, Yan X (2012) Novel natural food antimicro- Saldana Z, Xicohtencatl-Cortes J, Avelino F, Phillips AD, Kaper bials. Annu Rev Food Sci Technol 3:381–403 JB, Puente JL, Giron JA (2009) Synergistic role of curli and Kaplan JB (2011) Antibiotic-induced biofilm formation. Int J Artif cellulose in cell adherence and biofilm formation of attach- Organs 34:737–751 ing and effacing Escherichia coli and identification of Fis as Lei MG, Cue D, Roux CM, Dunman PM, Lee CY (2011) Rsp inhibits a negative regulator of curli. Environ Microbiol 11:992– attachment and biofilm formation by repressing fnbA in Staphy- 1006 lococcus aureus MW2. J Bacteriol 193:5231–5241 Sorroche FG, Spesia MB, Zorreguieta A, Giordano W (2012) A Lenz CA, Hew Ferstl CM, Vogel RF (2010) Sub-lethal stress effects on positive correlation between bacterial autoaggregation and bio- virulence gene expression in Enterococcus faecalis. Food Micro- film formation in native Sinorhizobium meliloti isolates from biol 27:317–326 Argentina. Appl Environ Microbiol 78:4092–4101 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression Van Houdt R, Michiels C (2010) Biofilm formation and the food -ΔΔCT data using real-time quantitative PCR and the 2 method. industry, a focus on the bacterial outer surface. J Appl Microbiol Methods 25:402–408 109:1117–1131
Annals of Microbiology – Springer Journals
Published: Nov 25, 2012
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