APMIS 122: 648–653
© 2013 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12210
Staphylococcal biofilm formation as affected by type acidulant ANTONIA NOSTRO,1 LUIGINA CELLINI,2 GIOVANNA GINESTRA,1 MANUELA D’ARRIGO,1 MARA DI GIULIO,2 ANDREANA MARINO,1 ANNA RITA BLANCO,3 ANGELO FAVALORO4,5 and GIUSEPPE BISIGNANO1 1 Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Universit a di Messina, Messina; Dipartimento di Farmacia, Universit a ‘G.d’Annunzio’, Chieti - Pescara; 3Research & Development S.I.F.I. SpA, Catania; 4Dipartimento di Scienze Biomediche e delle Immagini Morfologiche e Funzionali, Universit a di Messina, Messina; and 5IRCCS Centro Neurolesi ‘Bonino-Pulejo’, Messina, Italy 2
Nostro A, Cellini L, Ginestra G, D’Arrigo M, Giulio MD, Marino A, Blanco AR, Favaloro A, Bisignano G. Staphylococcal biofilm formation as affected by type acidulant. APMIS 2014; 122: 648–653. Staphylococcal growth and biofilm formation in culture medium where pH was lowered with weak organic (acetic and lactic) or strong inorganic (hydrochloric) acids were studied. The effects were evaluated by biomass measurements, cellsurface hydrophobicity, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). The results demonstrated that the inhibition was related to type of acidulant and pH value. At pH 5.0, the antibacterial effect was more pronounced in the presence of acetic acid (58–60% growth reduction) compared with that in the presence of lactic (7–16% growth reduction) and hydrochloric acids (23–24% reduction). The biofilm biomass of Staphylococcus aureus and Staphylococcus epidermidis was reduced by 92, 85, 63, and 93, 87, 81% after exposition to acetic, lactic, and hydrochloric acids, respectively. Increasing the pH from 5.0 to 6.0 resulted in a noticeable reduction in the effectiveness of acids. A minor cells hydrophobic character was also documented. The SEM and CLSM revealed a poorly structured and thinner biofilm compared with the dense and multilayered control. Acidic environment could have important implications for food-processing system to prevent bacterial colonization and control biofilm formation. The findings of this study lead to consider the rational use of the type of acid to achieve acidic environments. Key words: Biofilm; Staphylococcus aureus; Staphylococcus epidermidis; acidulant; pH. Antonia Nostro, Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Universit a di Messina, Viale S.S. Annunziata, 98168 Messina, Italy. e-mail: [email protected]
The biofilm formed on the surfaces or product contact surfaces is a potential source of contamination that may cause disease transmission with health hazards, or compromise product quality, with serious economic problems (1). Staphylococcus aureus and Staphylococcus epidermidis are pathogens able to produce biofilm on various materials and surfaces used in the biomedical, food, and industrial fields (2–4). Staphylococcal biofilm formation is a dynamic process that involves different mechanisms (5, 6). The initial adhesion phase is largely governed by physicochemical interactions between the bacteria and substrate and by environmental factors such as pH and temperature (7–10). Subsequently, the bacteria begin to synthesize intercellular adhesine and accumulate in multilayered cell clusters, so as Received 5 April 2013. Accepted 23 September 2013
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to be protected from the host immune system and from antimicrobial agents. Over the past two decades, research has focused on preventing and controlling biofilm formation. The acidification is an important method for controlling the bacterial growth (11). Weak organic acids have been found as safe antibacterial agents and they are among the most widely used antimicrobial food additives (12, 13) and pharmaceutical preservative agents (14). Organic acids are more effective than inorganic acids to inhibit the growth and metabolism of a wide range of pathogenic and spoilage microorganisms (15–17). Acetic acid has been reported to eradicate S. aureus from superficial wounds, skin lesions, and venous leg ulcers (18, 19). It was shown that bactericidal action of organic acids results from the undissociated form of acids that freely crosses the cell membrane,
STAPHYLOCOCCAL BIOFILM AND ACIDS
dissociates, and acidifies the cytoplasm, so lowering the internal pH (14). Some studies suggest that an acidic environment also has an impact on the biofilm formation (20, 21). In our previous work (22), we demonstrated the enhanced anti-biofilm activity of carvacrol against forming and established S. aureus and S. epidermidis biofilms at an acidic pH. The main focus of this study was to expand previous findings and evaluate the planktonic growth and the biofilm formation of S. aureus and S. epidermidis in culture medium where pH was reduced with either weak organic (acetic and lactic) or strong inorganic (hydrochloric) acids. Here, we sought to establish whether similar treatments could form the basis of novel strategy for staphylococcal biofilm controlling. To do this, biofilm biomass, cell-surface hydrophobicity, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM) analysis were carried out. MATERIALS AND METHODS Bacterial strains The bacteria used in this study were S. aureus 815 belonging to our private collection and the reference strains S. epidermidis ATCC 35984, a known slime producer. These strains have been selected for their previously wellcharacterized biofilm-related properties such as viscoelastic properties, icaA/icaD gene presence, slime production, capability of forming biofilm on polystyrene surface, hemolytic activity, and agr typing (23, 24). The strains were stored at 70 °C in MicrobanksTM (Prolab Diagnostics, Neston, UK); a single bead was removed from the cryovials and directly inoculated in Tryptic Soy Broth (TSB; Oxoid, Ltd, Basingstoke, London, UK). All reagents were purchased from Sigma (Milan, Italy) unless otherwise specified in the text.
Preparation of media Two weak organic acids namely Acetic acid (100%) (AA), L-Lactic acid (90%) (LA) and an inorganic strong acid namely Hydrochloric acid (HCl) (Merck, Milan, Italy) were used. Tryptic soy broth with 1% (w/v) glucose (TSBG, pH 7.2 0.05) was acidified by aseptically adding adequate amounts of AA (TSBGAA), LA (TSBGLA), and HCl (TSBGHCl) to the medium to achieve pH values of 5.0 0.03 and 6.0 0.05.
Planktonic growth curves The bacterial growth was tested in TSBGAA, TSBGLA, and TSBGHCl. The cultures grown overnight in 10 ml of TSBG were diluted to 5 9 105 CFU/mL in respective media at different pH values (5.0, 6.0). Aliquots of 2 mL were dispensed into glass test tubes and after incubation at 37 °C for 3, 6, and 24 h were controlled for planktonic bacterial growth by measuring the optical density at 492 nm using an spectrofotometer EIA reader (Bio-Rad
© 2013 APMIS. Published by John Wiley & Sons Ltd
Model 2550; Bio-Rad Laboratories, Inc., Richmond, CA, USA). Each assay was performed in quadruplicate and repeated at least three times. The growth control consisting of TSBG at pH 7.2 without acid was included.
Biofilm formation The bacterial biofilm-forming ability was tested in TSBGAA, TSBGLA, and TSBGHCl on polystyrene flatbottomed microtitre plates (Costar; Corning Inc., Corning, NY, USA) as previously reported (25) with some modifications. Briefly, the cultures were grown overnight in 10 mL of TSBG, diluted to 5 9 105 CFU/mL in acidified medium at different pH values (5.0 and 6.0) and 200 lL were dispensed into each well of 96-well polystyrene flatbottomed microtiter plates. After incubation of 3, 6, and 24 h at 37 °C, each well was washed three times with sterile phosphate-buffered saline (PBS; pH 7.4), dried, stained for 1 min with 0.1% safranin. The stained biofilms were resuspended in 30% acetic acid and optical density (OD492) was measured using a spectrophotometer EIA reader (Bio-Rad Laboratories, Inc.,). Each assay was performed in quadruplicate and repeated at least three times. The growth control consisting of TSBG at pH 7.2 without acid was included. The reduced percentage of biofilm formation in acidified medium was calculated by employing the ratio between the values of OD492nm in TSBGcontrol and TSBGwith acid by the following formula: 100- [(OD492 of TSBGwith acid/OD492 TSBGcontrol) 9 100].
Cell-surface hydrophobicity The S. aureus and S. epidermidis grown in TSBGLA were chosen for the cell-surface hydrophobicity measurements by a microbial adhesion to hydrocarbon (MATH) test as described by Martin et al. (26). Briefly, the cultures grown in TSBG medium were diluted to 5 9 105 CFU/mL in TSBGLA medium at two pH values (5.0 and 6.0). After incubation of 24 h at 37 °C, the cells were washed twice and suspended in sterile saline (0.85%), so that their optical density was 0.3 at 600 nm. The cell suspension (3 mL) was placed in tubes and 0.25 mL of toluene was added. The tubes were mixed uniformly in a vortex for 2 min and allowed to equilibrate at room temperature for 10 min. After toluene phase had been separated from the aqueous phase, the OD of the aqueous phase was determined spectrophotometrically at 600 nm. The hydrophobicity index (%HPBI) was calculated as: OD initial-OD final/OD initial 9 100.
Scanning electron microscopy The S. aureus and S. epidermidis biofilms formed at pH 5.0 in TSBGLA on transwell (Costar; Corning, Inc.) were chosen for SEM observations. The biofilms were fixed in glutaraldehyde 2% in 0.1 M PBS for 2 h at 4 °C and then post-fixed for 1 h at 4 °C in 1% osmium tetroxide in the same buffer. After thorough washing with PBS, samples were dehydrated in a series of ethanol solutions (30–100%). Specimens were mounted on aluminum stubs with conductive carbon cement, allowed to dry and then coated with a gold film. Samples were observed with an S-400 scanning electron microscope (Hitachi, Ltd., Tokyo, Japan).
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Confocal laser scanning microscopy The Staphylococcus aureus and S. epidermidis biofilms formed at pH 5.0 in TSBGLA on polystyrene plates (diameter, 15 mm, Costar; Corning, Inc.) were chosen for CLSM analysis. The biofilms were stained with the Live/ Dead Bach/Light bacterial viability kit (Molecular Probes Inc., Invitrogen, Eugene, OR, USA). The solution containing SYTO 9 and propidium iodide mixed in a ratio of 1:1 was added to the plates. The plates were incubated at room temperature for 15 min in the dark. After incubation, residual stain was removed. The sections were then analyzed and images acquired using a Zeiss LSM 5 DUO (Jena, Germany) confocal laser scanning microscope by META module. All images were digitalized at a resolution of 8 bits into an array of 2048 9 2048 pixels. Optical sections of fluorescent specimens were obtained using a HeNe laser (Zeiss) (wavelength = 543 nm) and an Argon laser (Zeiss) (wavelength = 458 nm) at a 1-min 2-s scanning speed with up to 8 averages; 1.50-lm-thick sections were obtained using a pinhole of 250. Contrast and brightness were established by examining the most brightly labeled pixels and choosing the settings that allowed clear visualization of the structural details while keeping the pixel intensity at its highest (ca. 200). Each image was acquired
AA pH 5.0 AA pH 6.0 Control
HCl pH 5.0 HCl pH 6.0
RESULTS AND DISCUSSION In this study, we have evaluated the growth and biofilm formation of S. aureus and S. epidermidis in culture media where pH was reduced with either weak organic or strong inorganic acids. The results demonstrated that the growth inhibition was related to acidulant type and pH value (Fig. 1A, B). At pH 5.0, except for the first detection at 3–6 h similar to the control, after prolonged incubation (6–24 h), the growth optical density was significantly lowered (5860%) in TSBGAA than that in TSBGLA (7–16%) and TSBGHCl (23–24%). Conversely, at pH 6.0, a
AA pH 5.0 AA pH 6.0
0.8
LA pH 5.0
HCl pH 5.0
LA pH 6.0
HCl pH 6.0
Control
0.6 0.4 0.2
0 1
3
6
24
B
0.8
Optical density (OD492)
Optical density (OD492)
test was employed to evaluate any significant differences between the values obtained in acidified medium compared with control. A p-value of LA>HCl. We can hypothesize that the poor biofilm formation in the presence of AA is due to the strong growth inhibition by the acid. However, this cannot be applied to the biofilm formed in the presence of LA and HCl as shown by a weak growth reduction (7–20%). Changes in the pH are known to have an impact on the expression of the regulatory systems in S. aureus (31, 32). This last cited work demonstrated the differences regarding how the S. aureus responded to the different acids. The ica genes were down-regulated by LA and HCl at pH 4.5, whereas the members of dltABCD operon were down-regulated by LA and up-regulated by HCl. These experimental evidences taken together could explain the inhibiting effects of the LA and HCl and also the major effect of LA compared to HCl. The ica genes are known to be central to intercellular adhesion as they encode for polysaccharide intercellular adhesin PIA. The dltABCD operon is known to regulate the addition of positively charged D-alanine residues that neutralize the net anionic charge of teicoic acids (33). A lesser degree of D-alanylation of teicoic acids determines a stronger negative cell-surface charge that leads to a lower interaction with surfaces and thereby compromises the initial bacterial adhesion (34). Hence, we verified the hydrophobic character of Staphylococci grown in TSBGLA by a microbial adhesion to solvents, test that measures an interplay of hydrophobic and electrostatic interactions (35). The results showed that S. aureus and S. epidermidis had a minor hydrophobicity index documented by a reduced affinity toward the toluene (Table 1). This could also interfere with the crucial step of adhesion and compromise the building of multilayered cell clusters. The SEM micrographs revealed a reduced biofilm of S. aureus and S. epidermidis grown in TSBGLA at pH 5.0 compared with the characteristic multilayered control grown in TSBG pH 7.2 (Fig. 3 top). The LA impairs the biofilm that results poorly structured, very thin, and arrested at the microcolony stage. These results were substantiated by the CLSM images that revealed a less organized
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biofilm formed in TSBGLA at pH 5.0 (Fig. 3 down). For S. aureus and S. epidermidis, this was thinner (2.76 9 0.31 lm and 4.05 0.15 lm thickness, respectively) than the dense and markedly viable biofilm formed in TSBG pH 7.2 (6.22 1.30 lm and 6.43 1.50 lm of thickness, respectively). CONCLUSION The findings of this study demonstrated that organic and inorganic acids reduced the biofilm formation of S. aureus and S. epidermidis. The inhibition was related to type of acidulant and pH value. The order of activity was acetic acid > lactic acid > hydrochloric acid. Acidic environment could have important implications for food-processing system to prevent the bacterial colonization and to control the biofilm formation. However, a rational use of type of acid to achieve acidic environments is needed.
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11. Raftari M, Jalilian FA, Abdulamir AS, Son R, Sekawi Z, Fatimah AB. Effect of organic acids on Escherichia coli O157:H7 and Staphylococcus aureus contaminated meat. Open Microbiol J 2009;3:121–7. 12. Brul S, Coote O. Preservative agents in foods. Mode of action and microbial resistance mechanisms. Int J Food Microbiol 1999;50:1–17. 13. Smulders FJ, Greer GG. Integrating microbial decontamination with organic acid in HACCP programmes from muscle foods: prospects and controversies. Int J Food Microbiol 1998;443:149–69. 14. Cherrington CA, Hinton M, Mead GC, Chopra I. Organic acids: chemistry, antibacterial activity and practical applications. Adv Microb Physiol 1991;32:87–108. 15. Castillo A, Lucia LM, Roberson DB, Stevenson TH, Mercado I, Acuff GR. Lactic acid sprays reduce bacterial pathogens on cold beef carcass surfaces and in subsequently produced ground beef. J Food Prot 2001;64:58–62. 16. Lund BM, Baird-Parker TC, Gould GW. The Microbiological Safety and Quality of Food. Gaithersburg, MD: Aspen Publishers Inc., 2000. 17. Samelis J, Sofos JN, Kain ML, Scanga JA, Belk KE, Smith GC. Organic acids and their salts as dipping solutions to control Listeria monocytogenes inoculated following processing of sliced pork bologna stored at 4 degrees C in vacuum packages. J Food Prot 2001;64:1722–9. 18. Nihei Y, Hasegawa T, Sato M. The efficacy of 3% acetic acid Vaseline for troublesome methicillin-resistant Staphylococcus aureus (MRSA) skin infection in a case of vesicular pemphigoid (in Japanese). Jpn J Dermatol 1993;103:27–32. 19. Hansson C, Faergemann J. The effect of antiseptic solutions on microorganisms in venous leg ulcers. Acta Derm Venereol 1995;75:31–3. 20. Dunne WM Jr, Burd EM. The effects of magnesium, calcium, EDTA, and pH on the in vitro adhesion of Staphylococcus epidermidis to plastic. Microbiol Immunol 1992;36:1019–27. 21. Zmantar T, Kouidhi B, Micadi H, Mahdouani K, Bakhrouf A. A Microtiter plate assay for Staphylococcus aureus biofilm quantification at various pH levels and hydrogen peroxide supplementation. New Microbiol 2010;33:137–45. 22. Nostro A, Cellini L, Zimbalatti V, Blanco AR, Marino A, Pizzimenti F, et al. Enhanced activity of carvacrol against biofilm of Staphylococcus aureus and Staphylococcus epidermidis in an acidic environment. APMIS 2012;120:967–73. 23. Blanco AR, Sudano-Roccaro A, Spoto GC, Nostro A, Rusciano D. Epigallocatechin gallate inhibits
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© 2013 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12210
Staphylococcal biofilm formation as affected by type acidulant ANTONIA NOSTRO,1 LUIGINA CELLINI,2 GIOVANNA GINESTRA,1 MANUELA D’ARRIGO,1 MARA DI GIULIO,2 ANDREANA MARINO,1 ANNA RITA BLANCO,3 ANGELO FAVALORO4,5 and GIUSEPPE BISIGNANO1 1 Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Universit a di Messina, Messina; Dipartimento di Farmacia, Universit a ‘G.d’Annunzio’, Chieti - Pescara; 3Research & Development S.I.F.I. SpA, Catania; 4Dipartimento di Scienze Biomediche e delle Immagini Morfologiche e Funzionali, Universit a di Messina, Messina; and 5IRCCS Centro Neurolesi ‘Bonino-Pulejo’, Messina, Italy 2
Nostro A, Cellini L, Ginestra G, D’Arrigo M, Giulio MD, Marino A, Blanco AR, Favaloro A, Bisignano G. Staphylococcal biofilm formation as affected by type acidulant. APMIS 2014; 122: 648–653. Staphylococcal growth and biofilm formation in culture medium where pH was lowered with weak organic (acetic and lactic) or strong inorganic (hydrochloric) acids were studied. The effects were evaluated by biomass measurements, cellsurface hydrophobicity, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). The results demonstrated that the inhibition was related to type of acidulant and pH value. At pH 5.0, the antibacterial effect was more pronounced in the presence of acetic acid (58–60% growth reduction) compared with that in the presence of lactic (7–16% growth reduction) and hydrochloric acids (23–24% reduction). The biofilm biomass of Staphylococcus aureus and Staphylococcus epidermidis was reduced by 92, 85, 63, and 93, 87, 81% after exposition to acetic, lactic, and hydrochloric acids, respectively. Increasing the pH from 5.0 to 6.0 resulted in a noticeable reduction in the effectiveness of acids. A minor cells hydrophobic character was also documented. The SEM and CLSM revealed a poorly structured and thinner biofilm compared with the dense and multilayered control. Acidic environment could have important implications for food-processing system to prevent bacterial colonization and control biofilm formation. The findings of this study lead to consider the rational use of the type of acid to achieve acidic environments. Key words: Biofilm; Staphylococcus aureus; Staphylococcus epidermidis; acidulant; pH. Antonia Nostro, Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Universit a di Messina, Viale S.S. Annunziata, 98168 Messina, Italy. e-mail: [email protected]
The biofilm formed on the surfaces or product contact surfaces is a potential source of contamination that may cause disease transmission with health hazards, or compromise product quality, with serious economic problems (1). Staphylococcus aureus and Staphylococcus epidermidis are pathogens able to produce biofilm on various materials and surfaces used in the biomedical, food, and industrial fields (2–4). Staphylococcal biofilm formation is a dynamic process that involves different mechanisms (5, 6). The initial adhesion phase is largely governed by physicochemical interactions between the bacteria and substrate and by environmental factors such as pH and temperature (7–10). Subsequently, the bacteria begin to synthesize intercellular adhesine and accumulate in multilayered cell clusters, so as Received 5 April 2013. Accepted 23 September 2013
648
to be protected from the host immune system and from antimicrobial agents. Over the past two decades, research has focused on preventing and controlling biofilm formation. The acidification is an important method for controlling the bacterial growth (11). Weak organic acids have been found as safe antibacterial agents and they are among the most widely used antimicrobial food additives (12, 13) and pharmaceutical preservative agents (14). Organic acids are more effective than inorganic acids to inhibit the growth and metabolism of a wide range of pathogenic and spoilage microorganisms (15–17). Acetic acid has been reported to eradicate S. aureus from superficial wounds, skin lesions, and venous leg ulcers (18, 19). It was shown that bactericidal action of organic acids results from the undissociated form of acids that freely crosses the cell membrane,
STAPHYLOCOCCAL BIOFILM AND ACIDS
dissociates, and acidifies the cytoplasm, so lowering the internal pH (14). Some studies suggest that an acidic environment also has an impact on the biofilm formation (20, 21). In our previous work (22), we demonstrated the enhanced anti-biofilm activity of carvacrol against forming and established S. aureus and S. epidermidis biofilms at an acidic pH. The main focus of this study was to expand previous findings and evaluate the planktonic growth and the biofilm formation of S. aureus and S. epidermidis in culture medium where pH was reduced with either weak organic (acetic and lactic) or strong inorganic (hydrochloric) acids. Here, we sought to establish whether similar treatments could form the basis of novel strategy for staphylococcal biofilm controlling. To do this, biofilm biomass, cell-surface hydrophobicity, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM) analysis were carried out. MATERIALS AND METHODS Bacterial strains The bacteria used in this study were S. aureus 815 belonging to our private collection and the reference strains S. epidermidis ATCC 35984, a known slime producer. These strains have been selected for their previously wellcharacterized biofilm-related properties such as viscoelastic properties, icaA/icaD gene presence, slime production, capability of forming biofilm on polystyrene surface, hemolytic activity, and agr typing (23, 24). The strains were stored at 70 °C in MicrobanksTM (Prolab Diagnostics, Neston, UK); a single bead was removed from the cryovials and directly inoculated in Tryptic Soy Broth (TSB; Oxoid, Ltd, Basingstoke, London, UK). All reagents were purchased from Sigma (Milan, Italy) unless otherwise specified in the text.
Preparation of media Two weak organic acids namely Acetic acid (100%) (AA), L-Lactic acid (90%) (LA) and an inorganic strong acid namely Hydrochloric acid (HCl) (Merck, Milan, Italy) were used. Tryptic soy broth with 1% (w/v) glucose (TSBG, pH 7.2 0.05) was acidified by aseptically adding adequate amounts of AA (TSBGAA), LA (TSBGLA), and HCl (TSBGHCl) to the medium to achieve pH values of 5.0 0.03 and 6.0 0.05.
Planktonic growth curves The bacterial growth was tested in TSBGAA, TSBGLA, and TSBGHCl. The cultures grown overnight in 10 ml of TSBG were diluted to 5 9 105 CFU/mL in respective media at different pH values (5.0, 6.0). Aliquots of 2 mL were dispensed into glass test tubes and after incubation at 37 °C for 3, 6, and 24 h were controlled for planktonic bacterial growth by measuring the optical density at 492 nm using an spectrofotometer EIA reader (Bio-Rad
© 2013 APMIS. Published by John Wiley & Sons Ltd
Model 2550; Bio-Rad Laboratories, Inc., Richmond, CA, USA). Each assay was performed in quadruplicate and repeated at least three times. The growth control consisting of TSBG at pH 7.2 without acid was included.
Biofilm formation The bacterial biofilm-forming ability was tested in TSBGAA, TSBGLA, and TSBGHCl on polystyrene flatbottomed microtitre plates (Costar; Corning Inc., Corning, NY, USA) as previously reported (25) with some modifications. Briefly, the cultures were grown overnight in 10 mL of TSBG, diluted to 5 9 105 CFU/mL in acidified medium at different pH values (5.0 and 6.0) and 200 lL were dispensed into each well of 96-well polystyrene flatbottomed microtiter plates. After incubation of 3, 6, and 24 h at 37 °C, each well was washed three times with sterile phosphate-buffered saline (PBS; pH 7.4), dried, stained for 1 min with 0.1% safranin. The stained biofilms were resuspended in 30% acetic acid and optical density (OD492) was measured using a spectrophotometer EIA reader (Bio-Rad Laboratories, Inc.,). Each assay was performed in quadruplicate and repeated at least three times. The growth control consisting of TSBG at pH 7.2 without acid was included. The reduced percentage of biofilm formation in acidified medium was calculated by employing the ratio between the values of OD492nm in TSBGcontrol and TSBGwith acid by the following formula: 100- [(OD492 of TSBGwith acid/OD492 TSBGcontrol) 9 100].
Cell-surface hydrophobicity The S. aureus and S. epidermidis grown in TSBGLA were chosen for the cell-surface hydrophobicity measurements by a microbial adhesion to hydrocarbon (MATH) test as described by Martin et al. (26). Briefly, the cultures grown in TSBG medium were diluted to 5 9 105 CFU/mL in TSBGLA medium at two pH values (5.0 and 6.0). After incubation of 24 h at 37 °C, the cells were washed twice and suspended in sterile saline (0.85%), so that their optical density was 0.3 at 600 nm. The cell suspension (3 mL) was placed in tubes and 0.25 mL of toluene was added. The tubes were mixed uniformly in a vortex for 2 min and allowed to equilibrate at room temperature for 10 min. After toluene phase had been separated from the aqueous phase, the OD of the aqueous phase was determined spectrophotometrically at 600 nm. The hydrophobicity index (%HPBI) was calculated as: OD initial-OD final/OD initial 9 100.
Scanning electron microscopy The S. aureus and S. epidermidis biofilms formed at pH 5.0 in TSBGLA on transwell (Costar; Corning, Inc.) were chosen for SEM observations. The biofilms were fixed in glutaraldehyde 2% in 0.1 M PBS for 2 h at 4 °C and then post-fixed for 1 h at 4 °C in 1% osmium tetroxide in the same buffer. After thorough washing with PBS, samples were dehydrated in a series of ethanol solutions (30–100%). Specimens were mounted on aluminum stubs with conductive carbon cement, allowed to dry and then coated with a gold film. Samples were observed with an S-400 scanning electron microscope (Hitachi, Ltd., Tokyo, Japan).
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Confocal laser scanning microscopy The Staphylococcus aureus and S. epidermidis biofilms formed at pH 5.0 in TSBGLA on polystyrene plates (diameter, 15 mm, Costar; Corning, Inc.) were chosen for CLSM analysis. The biofilms were stained with the Live/ Dead Bach/Light bacterial viability kit (Molecular Probes Inc., Invitrogen, Eugene, OR, USA). The solution containing SYTO 9 and propidium iodide mixed in a ratio of 1:1 was added to the plates. The plates were incubated at room temperature for 15 min in the dark. After incubation, residual stain was removed. The sections were then analyzed and images acquired using a Zeiss LSM 5 DUO (Jena, Germany) confocal laser scanning microscope by META module. All images were digitalized at a resolution of 8 bits into an array of 2048 9 2048 pixels. Optical sections of fluorescent specimens were obtained using a HeNe laser (Zeiss) (wavelength = 543 nm) and an Argon laser (Zeiss) (wavelength = 458 nm) at a 1-min 2-s scanning speed with up to 8 averages; 1.50-lm-thick sections were obtained using a pinhole of 250. Contrast and brightness were established by examining the most brightly labeled pixels and choosing the settings that allowed clear visualization of the structural details while keeping the pixel intensity at its highest (ca. 200). Each image was acquired
AA pH 5.0 AA pH 6.0 Control
HCl pH 5.0 HCl pH 6.0
RESULTS AND DISCUSSION In this study, we have evaluated the growth and biofilm formation of S. aureus and S. epidermidis in culture media where pH was reduced with either weak organic or strong inorganic acids. The results demonstrated that the growth inhibition was related to acidulant type and pH value (Fig. 1A, B). At pH 5.0, except for the first detection at 3–6 h similar to the control, after prolonged incubation (6–24 h), the growth optical density was significantly lowered (5860%) in TSBGAA than that in TSBGLA (7–16%) and TSBGHCl (23–24%). Conversely, at pH 6.0, a
AA pH 5.0 AA pH 6.0
0.8
LA pH 5.0
HCl pH 5.0
LA pH 6.0
HCl pH 6.0
Control
0.6 0.4 0.2
0 1
3
6
24
B
0.8
Optical density (OD492)
Optical density (OD492)
test was employed to evaluate any significant differences between the values obtained in acidified medium compared with control. A p-value of LA>HCl. We can hypothesize that the poor biofilm formation in the presence of AA is due to the strong growth inhibition by the acid. However, this cannot be applied to the biofilm formed in the presence of LA and HCl as shown by a weak growth reduction (7–20%). Changes in the pH are known to have an impact on the expression of the regulatory systems in S. aureus (31, 32). This last cited work demonstrated the differences regarding how the S. aureus responded to the different acids. The ica genes were down-regulated by LA and HCl at pH 4.5, whereas the members of dltABCD operon were down-regulated by LA and up-regulated by HCl. These experimental evidences taken together could explain the inhibiting effects of the LA and HCl and also the major effect of LA compared to HCl. The ica genes are known to be central to intercellular adhesion as they encode for polysaccharide intercellular adhesin PIA. The dltABCD operon is known to regulate the addition of positively charged D-alanine residues that neutralize the net anionic charge of teicoic acids (33). A lesser degree of D-alanylation of teicoic acids determines a stronger negative cell-surface charge that leads to a lower interaction with surfaces and thereby compromises the initial bacterial adhesion (34). Hence, we verified the hydrophobic character of Staphylococci grown in TSBGLA by a microbial adhesion to solvents, test that measures an interplay of hydrophobic and electrostatic interactions (35). The results showed that S. aureus and S. epidermidis had a minor hydrophobicity index documented by a reduced affinity toward the toluene (Table 1). This could also interfere with the crucial step of adhesion and compromise the building of multilayered cell clusters. The SEM micrographs revealed a reduced biofilm of S. aureus and S. epidermidis grown in TSBGLA at pH 5.0 compared with the characteristic multilayered control grown in TSBG pH 7.2 (Fig. 3 top). The LA impairs the biofilm that results poorly structured, very thin, and arrested at the microcolony stage. These results were substantiated by the CLSM images that revealed a less organized
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biofilm formed in TSBGLA at pH 5.0 (Fig. 3 down). For S. aureus and S. epidermidis, this was thinner (2.76 9 0.31 lm and 4.05 0.15 lm thickness, respectively) than the dense and markedly viable biofilm formed in TSBG pH 7.2 (6.22 1.30 lm and 6.43 1.50 lm of thickness, respectively). CONCLUSION The findings of this study demonstrated that organic and inorganic acids reduced the biofilm formation of S. aureus and S. epidermidis. The inhibition was related to type of acidulant and pH value. The order of activity was acetic acid > lactic acid > hydrochloric acid. Acidic environment could have important implications for food-processing system to prevent the bacterial colonization and to control the biofilm formation. However, a rational use of type of acid to achieve acidic environments is needed.
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