The Open Microbiology Journal


ISSN: 1874-2858 ― Volume 11, 2017

Ozone Sensitivity and Catalase Activity in Pigmented and Non-Pigmented Strains of Serratia Marcescens



José de Ondarza*
Department of Biological Sciences, Plattsburgh State University of New York, NY, USA

Abstract

Background:

Ozone exposure rapidly leads to bacterial death, making ozone an effective disinfectant in food industry and health care arena. However, microbial defenses may moderate this effect and play a role in the effective use of oxidizing agents for disinfection. Serratia marcescens is an opportunistic pathogen, expressing genes differentially during infection of a human host. A better understanding of regulatory systems that control expression of Serratia’s virulence genes and defenses is therefore valuable.

Objective:

Here, we investigated the role of pigmentation and catalase in Serratia marcescens on survival to ozone exposure.

Method:

Pigmented and non-pigmented strains of Serratia marcescens were cultured to exponential or stationary phase and exposed to 5 ppm of gaseous ozone for 2.5 – 10 minutes. Survival was calculated via plate counts. Catalase activity was measured photometrically and tolerance to hydrogen peroxide was assayed by disk-diffusion.

Results:

Exposure of S. marcescens to 5 ppm gaseous ozone kills > 90% of cells within 10 minutes in a time and concentration-dependent manner. Although pigmented Serratia (grown at 28°C) survived ozonation better than unpigmented Serratia (grown at 35°C), non-pigmented mutant strains of Serratia had similar ozone survival rates, catalase activity and H2O2 tolerance as wild type strains. Rather, ozone survival and catalase activity were elevated in 6 hour cultures compared to 48 hour cultures.

Conclusion:

Our studies did not bear out a role for prodigiosin in ozone survival. Rather, induction of oxidative stress responses during exponential growth increased both catalase activity and ozone survival in both pigmented and unpigmented S. marcescens.

Keywords: Catalase, oxyR, ozone, pigment, peroxide, prodigiosin, Serratia.


Article Information


Identifiers and Pagination:

Year: 2017
Volume: 11
First Page: 12
Last Page: 22
Publisher Id: TOMICROJ-11-12
DOI: 10.2174/1874285801711010012

Article History:

Received Date: 06/08/2016
Revision Received Date: 16/01/2017
Acceptance Date: 28/01/2017
Electronic publication date: 31/03/2017

© 2017 José de Ondarza

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of Biological Sciences, Plattsburgh State University of New York, NY, USA; Tel: 518-564-5156; Fax: 518-564-3175; E-mail: deondaj@plattsburgh.edu




INTRODUCTION

Ozone is a potent oxidizing agent that occurs naturally in the atmosphere at low (< 50 ppb) concentrations, but can rise to levels as high as 200 – 300 ppb in smog. At such levels, ozone becomes a significant health risk associated with respiratory irritation. At yet higher levels, its oxidizing potential can be life-threatening, but also serve as a useful disinfection agent with applications in food industry [1Pascual A, Llorca I, Canut A. Use of ozone in food industries for reducing the environmental impact of cleaning and disinfection activities. Trends Food Sci Technol 2007; 18(Suppl. 1): S29-35.
[http://dx.doi.org/10.1016/j.tifs.2006.10.006]
], water quality maintenance [2Gray NF, Percival SL, Yates MV, Williams D. Ozone disinfection. In: Microbiology of waterborne diseases. Jordan Hill, GBR: Academic Press: GBR 2013; pp. 599-615.], and in the health care industry [3Sharma M, Hudson JB. Ozone gas is an effective and practical antibacterial agent. Am J Infect Control 2008; 36(8): 559-63.
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]. The efficacy of ozone as a disinfecting agent has been reported to also vary significantly based on the concentration, time of exposure, presence of organic materials, and type of microbe exposed, with up to a 4-log reduction in pathogen counts on seeds reported at concentrations of 2000 ppm [5Trinetta V, Vaidya N, Linton R, Morgan M. A comparative study on the effectiveness of chlorine dioxide gas, ozone gas and e-beam irradiation treatments for inactivation of pathogens inoculated onto tomato, cantaloupe and lettuce seeds. Int J Food Microbiol 2011; 146(2): 203-6.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2011.02.014] [PMID: 21411164]
], although most treatment approaches use lower concentrations of 0.1 – 10 ppm with correspondingly lower reductions in microbial counts [6Yeoh WK, Ali A, Forney CF. Effects of ozone on major antioxidants and microbial populations of fresh-cut papaya. Postharvest Biol Technol 2014; 89: 56-8.
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]. A wide variety of pathogens respond differently to ozone disinfection, raising the question of whether sensitivity to ozone can be moderated by specific microbial defenses such as catalase, superoxide dismutase, or other antioxidant systems. Indeed, microbial responses such as elevation of SOD and catalase activity have been demonstrated in Listeria [8Fisher CW, Lee D, Dodge BA, Hamman KM, Robbins JB, Martin SE. Influence of catalase and superoxide dismutase on ozone inactivation of Listeria monocytogenes. Appl Environ Microbiol 2000; 66(4): 1405-9.
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] and E. coli [9Whiteside C, Hassan HM. Induction and inactivation of catalase and superoxide dismutase of Escherichia coli by ozone. Arch Biochem Biophys 1987; 257(2): 464-71.
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] in response to ozone exposure. Microbial responses to hydrogen peroxide exposure are better studied and reveal much of the underlying regulatory mechanisms, including induction of catalase, alkyl hydroperoxidase, and superoxide dismutase by oxidative stress via transactivator molecules such as oxyR [10Panmanee W, Hassett DJ. Differential roles of OxyR-controlled antioxidant enzymes alkyl hydroperoxide reductase (AhpCF) and catalase (KatB) in the protection of Pseudomonas aeruginosa against hydrogen peroxide in biofilm vs. planktonic culture. FEMS Microbiol Lett 2009; 295(2): 238-44.
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, 11Shanks RM, Stella NA, Kalivoda EJ, et al. A Serratia marcescens OxyR homolog mediates surface attachment and biofilm formation. J Bacteriol 2007; 189(20): 7262-72.
[http://dx.doi.org/10.1128/JB.00859-07] [PMID: 17675374]
], rpoS [12Liu X, Wu Y, Chen Y, et al. RpoS differentially affects the general stress response and biofilm formation in the endophytic Serratia plymuthica G3. Res Microbiol 2016; 167(3): 168-77.
[http://dx.doi.org/10.1016/j.resmic.2015.11.003] [PMID: 26671319]
] and soxRS [13Chiang SM, Schellhorn HE. Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria. Arch Biochem Biophys 2012; 525(2): 161-9.
[http://dx.doi.org/10.1016/j.abb.2012.02.007] [PMID: 22381957]
]. Alternatively, microbial pigmentation offers another potential defense against environmental challenges, including predation [14Matz C, Webb JS, Schupp PJ, et al. Marine biofilm bacteria evade eukaryotic predation by targeted chemical defense. PLoS One 2008; 3(7): e2744.
[http://dx.doi.org/10.1371/journal.pone.0002744] [PMID: 18648491]
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], competition (antibiotic effects), and potentially, oxygen radicals [16Itami C, Matsunaga H, Sawada T, Sakurai H, Kimura Y. Superoxide dismutase mimetic activities of metal complexes of L-2(2-pyridyl)-1-pyrroline-5-carboxylic acid (pyrimine). Biochem Biophys Res Commun 1993; 197(2): 536-41.
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-18Vaishnav P, Demain AL. Unexpected applications of secondary metabolites. Biotechnol Adv 2011; 29(2): 223-9.
[http://dx.doi.org/10.1016/j.biotechadv.2010.11.006] [PMID: 21130862]
]. Such pigments are often produced by bacteria as secondary metabolites via a quorum-sensing mechanism, including prodigiosin and violacein [19McClean KH, Winson MK, Fish L, et al. Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 1997; 143(Pt 12): 3703-11.
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[http://dx.doi.org/10.1111/j.1574-6976.2001.tb00583.x] [PMID: 11524130]
]. Whereas the purple pigment violacein can function as an anti-oxidant [22Konzen M, De Marco D, Cordova CA, Vieira TO, Antônio RV, Creczynski-Pasa TB. Antioxidant properties of violacein: possible relation on its biological function. Bioorg Med Chem 2006; 14(24): 8307-13.
[http://dx.doi.org/10.1016/j.bmc.2006.09.013] [PMID: 17011197]
], such a role has yet to be demonstrated for prodigiosin, a red pigment produced by Serratia marcescens. Serratia, a Gram-negative, ubiquitous microbe and opportunistic human pathogen, produces the red pigment prodigiosin in a temperature and quorum sensing-dependent manner, and possibly benefits from such pigments due to their antibiotic properties, although the exact function of these has not been completely elucidated [17Williamson NR, Fineran PC, Leeper FJ, Salmond GP. The biosynthesis and regulation of bacterial prodiginines. Nat Rev Microbiol 2006; 4(12): 887-99.
[http://dx.doi.org/10.1038/nrmicro1531] [PMID: 17109029]
]. The pigment (prodigiosin) is thought to be located in the cell wall of the bacteria and contributes to cell surface hydrophobicity [23Burger SR, Bennett JW. Droplet enrichment factors of pigmented and nonpigmented Serratia marcescens: possible selective function for prodigiosin. Appl Environ Microbiol 1985; 50(2): 487-90.
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, 24Rosenberg M, Blumberger Y, Judes H, Bar-Ness R, Rubinstein E, Mazor Y. Cell surface hydrophobicity of pigmented and nonpigmented clinical Serratia marcescens strains. Infect Immun 1986; 51(3): 932-5.
[PMID: 3512440]
], in a location where it could also conceivably act as a first layer of defense against external oxidative challenges. Alternately, it has been proposed that prodigiosin may act as an energy-spilling role, reducing ATP synthesis, oxidative metabolism and thus, reactive oxygen species generation [25Haddix PL, Jones S, Patel P, et al. Kinetic analysis of growth rate, ATP, and pigmentation suggests an energy-spilling function for the pigment prodigiosin of Serratia marcescens. J Bacteriol 2008; 190(22): 7453-63.
[http://dx.doi.org/10.1128/JB.00909-08] [PMID: 18805986]
]. We therefore sought to determine if pigmentation also offers a survival advantage and a defense against exposure to ozone. We show here that ozone kills Serratia marcescens in a time-dependent manner and that growth phase of the cultures, but not pigmentation or oxyR, affect survival to ozone exposure.

MATERIALS AND METHOD

Bacterial Strains

Serratia marcescens (wild type) was purchased from Presque Isle Cultures (Erie PA). All other Serratia marcescens strains were kindly provided by Dr. Robert Shanks (University of Pittsburgh) (Table 1).

Table 1
Strains used in this study.


Culture

All strains were initially grown in Tryptic Soy Broth (Hardy Diagnostics, Santa Maria CA) prior to plating on MacConkey agar (Becton Dickinson, Franklin Lakes NJ) for ozone exposure. Wild-type S. marcescens was cultured for 48h at either 30°C (red-pigmented, SM-R) or 37°C, a temperature at which prodigiosin is not produced (SM-W). S. marcescens strains SM-01, SM-02, SM-04, SM-05, and SM-06 were cultured 6h (exponential phase) or 48h (stationary phase) at 30°C, yielding pigmentation in wild-type and oxyR mutants but not the pigA deletion mutants. Pigmentation level was assayed as the ratio of light absorbance at 535 nm/600 nm. 1 ml of each 48h cell culture was centrifuged at 13,000g to pellet cells, supernatant discarded, and pigment extracted in 1 ml acid-alcohol (95% isopropanol + 1% 2N HCl). A600 was measured for 500 µl of cell culture resuspended in 2 ml saline; A535 was measured for 500µl pigment extract resuspended in 2 ml dH2O.

Ozone Exposure

Growing (6h) or stationary (48h) cultures of S. marcescens were diluted in TSB to an Absorbance of 0.100 at 600 nm, serial-diluted to 10-4 or 10-5 in 0.9% saline, and spread-plated onto 125 mm MacConkey agar plates. Agar plates were then exposed to 5 or 10 ppm ozone inside a 42L plexiglass chamber. Ozone was generated with an OZX-300 Ozone generator (Enaly Co, Shanghai, China) and continuously monitored with a Model 106-L Ozone Monitor (2B Technologies, Boulder CO). Concentrations were maintained to within 0.5 ppm. Exposure times ranged from 2.5 minutes to 20 minutes. Agar plates were subsequently incubated for 24 - 48h at room temperature, colonies counted, and survival calculated as a percentage of unexposed controls.

Pre-Exposure of Serratia to H2O2

Exponentially growing 5 ml TSB cultures of S. marcescens were subjected to oxidative stress by pre-exposure to low (200 µM) or high (20 mM) levels of H2O2 for 30 min prior to ozone exposure and catalase measurements.

H2O2 Disk Diffusion Assay

Sensitivity of S. marcescens to H2O2 was determined by disk-diffusion assay. Pour plates were prepared by mixing 100μl of a 0.500 Absorbance (600 nm) culture broth of each test strain with 2 ml of melted (45°C) Top Agar (50% Trypticase soy agar/50% Tryticase soy broth) and pouring it over bottom agar (10 ml TSA in 48 mm sterile Petri plates). Once solidified, sterile 6 mm blank paper disks were placed on the agar surface and 10 µl of 30% H2O2 (Sigma-Aldrich) was added to each disk. Plates were incubated for 24h at either 28°C or 37°C and zones of inhibition measured. Each assay was conducted at least 3 separate times on different days.

Catalase Assay

Catalase activity was assayed following the methods of Beers [26Beers RF Jr, Sizer IW. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 1952; 195(1): 133-40.
[PMID: 14938361]
]. In brief, 1 ml of each SM broth culture was pelleted by centrifugation at 6,000 x g, washed and resuspended in 2 ml of 0.9% saline, and sonicated at 40% power twelve times (30s on/30s off) on ice. Cell lysates were centrifuged for 20 min at 12000 x g to pellet cell debris, and supernatants were collected. 20µl of each lysate, supernatant, and pellet (resuspended in 1 ml saline) were then added to 50 mM phosphate buffer (pH 7) containing 30 mM H2O2. Occasionally, 100 μl lysate samples were heated to either 50°C or 100°C for 20 minutes before assaying. Catalase activity was measured by following the change in absorbance at 240 nm in an Ocean Optics 4400 UV/Vis spectrometer and calculated as (ΔA240/min)/(43.6/Mol/min * sample volume * 0.001). Catalase activity was normalized to sample protein concentration when possible using Bradford protein assay.

Statistical Analysis

Pairwise comparisons were performed using Student’s t-test, while multiple comparisons were conducted using one-way ANOVA with GraphPad Prism software.

RESULTS

Survival to Ozone Exposure

Exposure of 48 h cultures of Serratia marcescens to ozone showed a time-dependent and concentration-dependent reduction in viable cell numbers for both pigmented and unpigmented strains. Initially, brief 30 second exposures to a range of ozone concentrations were tested to determine effective concentrations for short-term (5 – 30 minute) exposures. Based on these results (data not shown), concentrations of 5 and 10 ppm were selected. Low levels of ozone (5 ppm) significantly reduced bacterial numbers over a 10 minute time period, affecting non-pigmented S. marcescens (SM-W) more than pigmented strains (SM-R) (Fig. 1). This effect was significant at 2.5 min exposure (P<0.05), but also pronounced at 5 and 7.5 min. Similarly, ozonation at 10 ppm reduced SM-01 cell numbers, but at a faster rate (10-fold reduction in 5 min). Pigmentation in 48h broth cultures of SM-R (cultured at 30°C) was robust, attaining an Absorbance ratio (A534/A600) of 0.472, whereas the same strain grown at 37°C exhibited no pigmentation (A535/A600 ratio = 0.005). To clarify whether pigmentation, or some other change brought about by lower incubation temperature, was responsible for the lower sensitivity to ozone, this experiment was repeated using two wild-type S. marcescens strains (SM-01 and SM-04 Nima) and their pigA deletion mutants (SM-02 and SM-05, respectively), with incubation conditions maintained at 30°C. Both mutant strains exhibited similar survival following 5 ppm ozone exposure as their pigmented counterparts (Fig. 2), strongly suggesting that pigmentation may not be responsible for the observed difference in sensitivity to ozone.

Fig. (1)
Survival of Serratia marcescens after exposure to 2.5 – 10 min of gaseous ozone (5 ppm). Pigmented SM-R (open squares) survived ozone exposure better than unpigmented SM-W (filled diamonds); * significantly different (P < 0.05).


Fig. (2)
Survival of wild type and mutant S. marcescens strains following ozone (5 ppm) exposure. S. marcescens strains were incubated for either 6h or 48h prior to ozonation. No significant differences were observed among strains in stationary phase (48h) or among strains in exponential phase (6h), but survival was significantly greater (P < 0.05) for exponential-phase cultures compared to stationary-phase cultures.


Sensitivity to Hydrogen Peroxide Exposure

To investigate whether expression of anti-oxidant enzymes such as catalase mediate this protection against ozone exposure, SM-R and SM-W were suspended in top agar and pour-plated onto a bottom agar base prior to incubation in the presence of 10µl of 30% H2O2 in a disk-diffusion assay. Although SM-R had slightly smaller inhibition zones when assayed at pigment-producing temperature (28°C) than SM-W assayed at 37°C (Fig. 3), this difference was not significant. As well, SM-W also showed slightly higher (P > 0.05) resistance to H2O2 at 28°C, and greater resistance to H2O2 than SM-R at each assay temperature. This suggests that SM-W has better peroxide defenses (and potentially) catalase activity. A similar pattern was observed when comparing the SM-04 (Nima) strain to its pigA deletion counterpart (SM-05), with pigmented SM-04 having a similar, though slightly smaller, inhibition zone than unpigmented SM-05 at 28°C, or SM-04 assayed at 37°C (unpigmented). In comparison, both E. coli (known to have multiple catalases) and Streptococcus lactis (lacking catalase) have significantly smaller inhibition zones at 37°C (Fig. 3), as does an oxyR mutant of Serratia (SM-06).

Fig. (3)
Effect of assay temperature on H2O2 sensitivity (Zone of inhibition diameter) for E. coli, Streptococcus lactis (SL), and S. marcescens strains SM-01, SM-02, SM-06 (all cultured at 30°C) and SM wild type cultured at either 28°C (SM-R) or 37°C (SM-W) before exposure to 10µl H2O2. Agar plates + H2O2-impregnated disks were incubated for 24h at 28°C or 37°C. Incubation at pigment-producing temperature (28°C) slightly increased SM resistance to H2O2 exposure, whereas E. coli, catalase-lacking Streptococcus lactis (SL), and the oxyR mutant of Serratia (SM-06) are more sensitive (P < 0.05) at this temperature. * significantly different (28°C vs 37°C).


Catalase Activity in Serratia Strains

To assess catalase activity directly, H2O2 degradation was measured spectrophotometrically. Consistent with the disk diffusion data, SM-W (grown at 37°C) exhibited slightly higher catalase activity than SM-R (grown at 28°C) (Table 2). Catalase activity was detected in the cell lysate following sonication, in the soluble fraction (supernatant) of the lysate following centrifugation, in heated (50°C) lysate and in intact cells, but not in the pelleted cell debris or in boiled supernatant (Fig. 4). Heating of cell lysates to 50°C reduced catalase activity by 3% - 15% in all strains examined, whereas heating to 75°C reduced activity by 84% - 90% and boiling reduced activity essentially to background levels. Catalase activity was also shown to be slightly greater in the non-pigmented SM-02 pigA mutant than in the wild type strain SM-01.

Table 2
Catalase activity of S. marcescens cell lysates following centrifugation to separate cell debris (pellet) from soluble components (supernatant). Activity was determined in stationary phase cells (48h cultures, N = 6) or exponential phase cells (6h cultures, N = 7) as units/ml as well as normalized to sample protein concentrations (U/mg). Protein concentrations were not determined for pelleted debris due to catalase activities indistinguishable from background.


Fig. (4)
Catalase activity (U/mg) of pigmented (red) and unpigmented (white) Serratia marcescens cell lysates, normalized to protein concentrations. Heating of lysates to 50°C did not affect catalase activity, but activity was significantly lower in lysates heated to 75°C or 100°C (P < 0.05). For temperature stability assays, only cell lysates were used.


Sensitivity to Ozone and H2O2, and Catalase Activity, of oxyR Mutant S. Marcescens

To further investigate the role of oxidative stress defenses in ozone sensitivity and catalase activity, an oxyR mutant strain [11Shanks RM, Stella NA, Kalivoda EJ, et al. A Serratia marcescens OxyR homolog mediates surface attachment and biofilm formation. J Bacteriol 2007; 189(20): 7262-72.
[http://dx.doi.org/10.1128/JB.00859-07] [PMID: 17675374]
] was assayed. OxyR serves as a sensor of oxidative stress in many bacteria and regulates expression of catalase [13Chiang SM, Schellhorn HE. Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria. Arch Biochem Biophys 2012; 525(2): 161-9.
[http://dx.doi.org/10.1016/j.abb.2012.02.007] [PMID: 22381957]
]. 48h cultures of SM-06 (OxyR mutant) had similar ozone survival (Fig. 2) and catalase activity (Table 2) as the wild-type (SM-01) strain even while maintaining their greater sensitivity to H2O2 exposure in a disk-diffusion assay as previously established [11Shanks RM, Stella NA, Kalivoda EJ, et al. A Serratia marcescens OxyR homolog mediates surface attachment and biofilm formation. J Bacteriol 2007; 189(20): 7262-72.
[http://dx.doi.org/10.1128/JB.00859-07] [PMID: 17675374]
]. Interestingly, the oxyR mutant exhibited a lower sensitivity to H2O2 at 37°C, much like E. coli and S. lactis (Fig. 3) and unlike Serratia strains SM-01 and SM-02.

Effect of H2O2 Pre-Exposure on Ozone Sensitivity and Catalase Activity

Since the putative function of oxyR is to up-regulate microbial defenses against oxidative stress, we then repeated the ozone and catalase assays in Serratia strains which were cultured for 6h (exponential phase) and then pre-exposed to oxidative stress (200 μM or 20 mM H2O2). Catalase activity in 6h cultures was significantly greater than that of 48h cultures in SM-01 (pigmented) and SM-02 (non-pigmented), but not in SM-06 (oxyR mutant) strains of S. marcescens (Table 2). However, no significant differences were observed between catalase activity of H2O2-pre-exposed cultures and controls (P > 0.48). We also examined the effect of H2O2 pre-exposure in 6h cultures on S. marcescens sensitivity to ozone exposure. All strains of Serratia tested after 6h of incubation (SM-01, 02 and 06) exhibited a significantly greater survival to ozonation at 5 ppm than any stationary phase (48h culture) Serratia strains (Fig. 2). Pre-exposure to H2O2 had a slight but not significant effect on ozone survival, with slightly better 5 min survival rates for 6h cultures pre-exposed to 50mM H2O2.

DISCUSSION

Effect of Ozone Exposure on S. Marcescens

Ozone is a potent and highly reactive molecule which can rapidly induce cellular damage in bacteria as well as humans. Its potent antibacterial action has led to its resurgence as a disinfectant in industrial, medical and food industry settings [2Gray NF, Percival SL, Yates MV, Williams D. Ozone disinfection. In: Microbiology of waterborne diseases. Jordan Hill, GBR: Academic Press: GBR 2013; pp. 599-615.]. Because it is naturally occurring, we thought it plausible that bacterial defenses against oxidizing agents will protect them from ozone disinfection. Here, we initially showed that continuous exposure to 5 – 10 ppm of gaseous ozone kills S. marcescens in a time and concentration-dependent manner, and that these reductions are in line with reported ozone killing efficiencies in fruits and vegetables [6Yeoh WK, Ali A, Forney CF. Effects of ozone on major antioxidants and microbial populations of fresh-cut papaya. Postharvest Biol Technol 2014; 89: 56-8.
[http://dx.doi.org/10.1016/j.postharvbio.2013.11.006]
, 27Alwi NA, Ali A. Reduction of Escherichia coli O157, Listeria monocytogenes and Salmonella enterica sv. Typhimurium populations on fresh-cut bell pepper using gaseous ozone. Food Contr 2014; 46: 304-11.
[http://dx.doi.org/10.1016/j.foodcont.2014.05.037]
, 28Bermúdez-Aguirre D, Barbosa-Cánovas GV. Disinfection of selected vegetables under nonthermal treatments: Chlorine, acid citric, ultraviolet light and ozone. Food Contr 2013; 29(1): 82-90.
[http://dx.doi.org/10.1016/j.foodcont.2012.05.073]
] where microbes attached to food surfaces were effectively killed by 0.1 – 9.2 ppm of ozone. Although up to 4-log reductions were observed with dry seed surfaces [5Trinetta V, Vaidya N, Linton R, Morgan M. A comparative study on the effectiveness of chlorine dioxide gas, ozone gas and e-beam irradiation treatments for inactivation of pathogens inoculated onto tomato, cantaloupe and lettuce seeds. Int J Food Microbiol 2011; 146(2): 203-6.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2011.02.014] [PMID: 21411164]
], the above-mentioned studies reported more typical log10 reductions of 0.22 – 2.89 for pathogens on moist food surfaces, depending on bacterial strain and exposure time. In the present study, Serratia cells were attached to a moist (agar) surface, and exposure times were similar or shorter (2.5 to 10 min vs. 5 min to 6 h) than in previous studies, but in general we attained a similar 1 – 2 log reduction in 10 min at 5 ppm (Ct value = 50 ppm.min) as with Salmonella and E. coli on bell peppers treated for 30 min at 1 ppm [27Alwi NA, Ali A. Reduction of Escherichia coli O157, Listeria monocytogenes and Salmonella enterica sv. Typhimurium populations on fresh-cut bell pepper using gaseous ozone. Food Contr 2014; 46: 304-11.
[http://dx.doi.org/10.1016/j.foodcont.2014.05.037]
], E. coli on carrots and lettuce treated for 15 min at 5 ppm [28Bermúdez-Aguirre D, Barbosa-Cánovas GV. Disinfection of selected vegetables under nonthermal treatments: Chlorine, acid citric, ultraviolet light and ozone. Food Contr 2013; 29(1): 82-90.
[http://dx.doi.org/10.1016/j.foodcont.2012.05.073]
], and coliforms on papaya treated for 20 min at 9.2 ppm [6Yeoh WK, Ali A, Forney CF. Effects of ozone on major antioxidants and microbial populations of fresh-cut papaya. Postharvest Biol Technol 2014; 89: 56-8.
[http://dx.doi.org/10.1016/j.postharvbio.2013.11.006]
]. Ozone is thought to disrupt bacterial cell envelopes, leading to cell death by cytoplasmic leakage and cell lysis [1Pascual A, Llorca I, Canut A. Use of ozone in food industries for reducing the environmental impact of cleaning and disinfection activities. Trends Food Sci Technol 2007; 18(Suppl. 1): S29-35.
[http://dx.doi.org/10.1016/j.tifs.2006.10.006]
, 27Alwi NA, Ali A. Reduction of Escherichia coli O157, Listeria monocytogenes and Salmonella enterica sv. Typhimurium populations on fresh-cut bell pepper using gaseous ozone. Food Contr 2014; 46: 304-11.
[http://dx.doi.org/10.1016/j.foodcont.2014.05.037]
].

Effect of Pigmentation on Survival to Ozone Exposure

Unlike most other coliforms, Serratia marcescens is capable of producing the red pigment prodigiosin. Expression of prodigiosin is under complex regulatory control; its genes are part of the pig operon (pig for pigment) which allows pigment production to be regulated in a temperature and quorum-sensing-dependent manner [17Williamson NR, Fineran PC, Leeper FJ, Salmond GP. The biosynthesis and regulation of bacterial prodiginines. Nat Rev Microbiol 2006; 4(12): 887-99.
[http://dx.doi.org/10.1038/nrmicro1531] [PMID: 17109029]
, 20Van Houdt R, Givskov M, Michiels CW. Quorum sensing in Serratia. FEMS Microbiol Rev 2007; 31(4): 407-24.
[http://dx.doi.org/10.1111/j.1574-6976.2007.00071.x] [PMID: 17459113]
]. Our observation of greater survival of pigmented Serratia cells to ozone exposure than cells of the same strain that are unpigmented (when cultured at higher temperatures) could therefore be explained either by the presence of pigment or by differential expression of other protective factors at the lower temperature. Cultures of wild-type SM grown at 30°C for 48h were intensely pigmented; this pigmentation is presumably located in the bacterial wall [17Williamson NR, Fineran PC, Leeper FJ, Salmond GP. The biosynthesis and regulation of bacterial prodiginines. Nat Rev Microbiol 2006; 4(12): 887-99.
[http://dx.doi.org/10.1038/nrmicro1531] [PMID: 17109029]
] and thus present in single cells of pigmented, but not unpigmented, cultures. Such cells consistently survived ozone exposure better than unpigmented cells of the same strain cultured at 37°C (Fig. 1). However, neither of the pigA-mutant strains of Serratia differed from their isogenic pigmented strains in term of ozone survival, and it is therefore likely that a temperature-induced change in protective genes could be responsible. The transition from environmental temperatures to that of an animal host is known to induce significant alterations in gene expression in many pathogens, including dimorphic fungi and environmental bacteria such as Listeria [29Gründling A, Burrack LS, Bouwer HG, Higgins DE. Listeria monocytogenes regulates flagellar motility gene expression through MogR, a transcriptional repressor required for virulence. Proc Natl Acad Sci USA 2004; 101(33): 12318-23.
[http://dx.doi.org/10.1073/pnas.0404924101] [PMID: 15302931]
], Pseudomonas aeruginosa [30Grosso-Becerra MV, Croda-García G, Merino E, Servín-González L, Mojica-Espinosa R, Soberón-Chávez G. Regulation of Pseudomonas aeruginosa virulence factors by two novel RNA thermometers. Proc Natl Acad Sci USA 2014; 111(43): 15562-7.
[http://dx.doi.org/10.1073/pnas.1402536111] [PMID: 25313031]
], and Serratia, where porin production [31Begic S, Worobec EA. Regulation of Serratia marcescens ompF and ompC porin genes in response to osmotic stress, salicylate, temperature and pH. Microbiology 2006; 152(Pt 2): 485-91.
[http://dx.doi.org/10.1099/mic.0.28428-0] [PMID: 16436436]
], pigmentation, and possibly catalase expression [32Crichton PV, Lazarus AS. The relationship between prodigiosin production and catalase activity. J Bacteriol 1948; 56(3): 375-7.
[PMID: 16561583]
] are changed.

Possible Role of Catalase in Ozone Resistance

Catalase expression is of particular interest, as catalase activity is affected by ozone exposure [8Fisher CW, Lee D, Dodge BA, Hamman KM, Robbins JB, Martin SE. Influence of catalase and superoxide dismutase on ozone inactivation of Listeria monocytogenes. Appl Environ Microbiol 2000; 66(4): 1405-9.
[http://dx.doi.org/10.1128/AEM.66.4.1405-1409.2000] [PMID: 10742219]
, 33Lee YK, Mok Kim S, Han S. Ozone-induced inactivation of antioxidant enzymes. Biochimie 2003; 85(10): 947-52.
[http://dx.doi.org/10.1016/j.biochi.2003.09.012] [PMID: 14644549]
], and we therefore examined more closely the role of catalase in Serratia. Genes for monofunctional (HPII; katE, 478 aa) and bifunctional (HPI; katG, 731 aa) catalase have been sequenced in Serratia [34Chung WC, Chen LL, Lo WS, Kuo PA, Tu J, Kuo CH. Complete genome sequence of Serratia marcescens WW4. Genome Announc 2013; 1(2): e0012613.
[http://dx.doi.org/10.1128/genomeA.00126-13] [PMID: 23558532]
, 35Daligault HE, Davenport KW, Minogue TD, et al. Genome assembly of Serratia marcescens type Strain ATCC 13880. Genome Announc 2014; 2(5): e00967-14.
[PMID: 25291774]
]. Presumably, then, both constitutive and inducible forms of catalase operate in Serratia. Direct measurement of catalase activity show similar levels in pigmented (SM-R, SM-01) and non-pigmented (SM-W and SM-02) strains cultured for 2 or more days (Table 2). This catalase activity is likely due to the monofunctional catalase (HPII), which, at least in E. coli, is increasingly expressed as cells transition into stationary phase [36Loewen PC, Switala J, Triggs-Raine BL. Catalases HPI and HPII in Escherichia coli are induced independently. Arch Biochem Biophys 1985; 243(1): 144-9.
[http://dx.doi.org/10.1016/0003-9861(85)90782-9] [PMID: 3904630]
]. This is consistent with detection of monofunctional catalase activity in S. marcescens [37Switala J, Triggs-Raine BL, Loewen PC. Homology among bacterial catalase genes. Can J Microbiol 1990; 36(10): 728-31.
[http://dx.doi.org/10.1139/m90-123] [PMID: 2253114]
, 38Zeng HW, Cai YJ, Liao XR, Zhang F, Zhang DB. Production, characterization, cloning and sequence analysis of a monofunctional catalase from Serratia marcescens SYBC08. J Basic Microbiol 2011; 51(2): 205-14.
[http://dx.doi.org/10.1002/jobm.201000147] [PMID: 21077118]
]. The catalase described by Zeng [38Zeng HW, Cai YJ, Liao XR, Zhang F, Zhang DB. Production, characterization, cloning and sequence analysis of a monofunctional catalase from Serratia marcescens SYBC08. J Basic Microbiol 2011; 51(2): 205-14.
[http://dx.doi.org/10.1002/jobm.201000147] [PMID: 21077118]
] is also relatively stable up to 70°C, consistent with our data (Fig. 2). The HPI form of catalase, in contrast, is a bifunctional catalase that is encoded by katG and is under control of the OxyR regulon in E. coli [13Chiang SM, Schellhorn HE. Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria. Arch Biochem Biophys 2012; 525(2): 161-9.
[http://dx.doi.org/10.1016/j.abb.2012.02.007] [PMID: 22381957]
, 39Brown A, Lo D, Sharpe C, et al. Increased intracellular hydrogen peroxide observed in Escherichia coli in the presence of two classes of antibiotics. J Exp Microbiol Immunol 2012; 16: 17-23., 40Eason MM, Fan X. The role and regulation of catalase in respiratory tract opportunistic bacterial pathogens. Microb Pathog 2014; 74: 50-8.
[http://dx.doi.org/10.1016/j.micpath.2014.07.002] [PMID: 25109939]
], an operon which also functions in Serratia marcescens [11Shanks RM, Stella NA, Kalivoda EJ, et al. A Serratia marcescens OxyR homolog mediates surface attachment and biofilm formation. J Bacteriol 2007; 189(20): 7262-72.
[http://dx.doi.org/10.1128/JB.00859-07] [PMID: 17675374]
]. Although this enzyme was not directly detected in Serratia in either mid-log or stationary phase cells [37Switala J, Triggs-Raine BL, Loewen PC. Homology among bacterial catalase genes. Can J Microbiol 1990; 36(10): 728-31.
[http://dx.doi.org/10.1139/m90-123] [PMID: 2253114]
], exposure of Serratia cells to 50 mM H2O2 did indeed induce katG expression dramatically in the recently described high oxidative stress-tolerant isolate Serratia sp. LCN16, a response which was absent in oxyR mutants [41Vicente CS, Nascimento FX, Ikuyo Y, Cock PJ, Mota M, Hasegawa K. The genome and genetics of a high oxidative stress tolerant Serratia sp. LCN16 isolated from the plant parasitic nematode Bursaphelenchus xylophilus. BMC Genomics 2016; 17(1): 301.
[http://dx.doi.org/10.1186/s12864-016-2626-1] [PMID: 27108223]
].

Response of S. Marcescens to Oxidative Stress (H2O2 Exposure)

Here, we were unable to observe a significant increase in catalase activity following low (200 μM) or high (20 mM) level H2O2 pretreatments; rather, we observed a significantly higher catalase activity in 6h cultures of wild-type, but not oxyR mutant, S. marcescens as compared to 48h cultures (stationary). It is therefore possible that rapid early growth of Serratia generates sufficient oxidative stress through aerobic growth to activate a stress response via oxyR. This can also explain the reduced resistance of oxyR mutants in H2O2 disk-diffusion assays [11Shanks RM, Stella NA, Kalivoda EJ, et al. A Serratia marcescens OxyR homolog mediates surface attachment and biofilm formation. J Bacteriol 2007; 189(20): 7262-72.
[http://dx.doi.org/10.1128/JB.00859-07] [PMID: 17675374]
], where the lack of an oxidative stress response makes these cells sensitive to normally sub-lethal doses of hydrogen peroxide. This oxyR-mediated protective response may well be mediated by katG. During aerobic growth of Bradyrhizobium japonicum [42Panek HR, OBrian MR. KatG is the primary detoxifier of hydrogen peroxide produced by aerobic metabolism in Bradyrhizobium japonicum. J Bacteriol 2004; 186(23): 7874-80.
[http://dx.doi.org/10.1128/JB.186.23.7874-7880.2004] [PMID: 15547258]
] katG has even been shown to be the main way to remove endogenously generated H2O2, albeit without oxyR.

Protective Mechanisms to Oxidative Stress

The mechanism of action of ozone on bacterial cells involves oxidation of cellular components, in part through generation of reactive oxygen species such as O˙, OH˙, and hydrogen peroxide. These reactive oxygen species in turn combine with cellular lipids and proteins, causing membrane, enzyme inactivation and cell lysis [1Pascual A, Llorca I, Canut A. Use of ozone in food industries for reducing the environmental impact of cleaning and disinfection activities. Trends Food Sci Technol 2007; 18(Suppl. 1): S29-35.
[http://dx.doi.org/10.1016/j.tifs.2006.10.006]
, 27Alwi NA, Ali A. Reduction of Escherichia coli O157, Listeria monocytogenes and Salmonella enterica sv. Typhimurium populations on fresh-cut bell pepper using gaseous ozone. Food Contr 2014; 46: 304-11.
[http://dx.doi.org/10.1016/j.foodcont.2014.05.037]
, 33Lee YK, Mok Kim S, Han S. Ozone-induced inactivation of antioxidant enzymes. Biochimie 2003; 85(10): 947-52.
[http://dx.doi.org/10.1016/j.biochi.2003.09.012] [PMID: 14644549]
]. Since hydrogen peroxide is a byproduct of ozone reactions with cells, peroxidases are reasonable means of cellular defenses against ozone [8Fisher CW, Lee D, Dodge BA, Hamman KM, Robbins JB, Martin SE. Influence of catalase and superoxide dismutase on ozone inactivation of Listeria monocytogenes. Appl Environ Microbiol 2000; 66(4): 1405-9.
[http://dx.doi.org/10.1128/AEM.66.4.1405-1409.2000] [PMID: 10742219]
, 33Lee YK, Mok Kim S, Han S. Ozone-induced inactivation of antioxidant enzymes. Biochimie 2003; 85(10): 947-52.
[http://dx.doi.org/10.1016/j.biochi.2003.09.012] [PMID: 14644549]
]. Indeed, defensive enzymes such as catalase have been shown to be induced by ozone exposure [9Whiteside C, Hassan HM. Induction and inactivation of catalase and superoxide dismutase of Escherichia coli by ozone. Arch Biochem Biophys 1987; 257(2): 464-71.
[http://dx.doi.org/10.1016/0003-9861(87)90591-1] [PMID: 3310892]
]. While our observation that exponentially growing cells have both greater ozone survival and catalase activity support a protective role of catalase, other mechanisms are certainly at work since survival of the oxyR mutant (SM-06) strain to ozone exposure was similar to that of the wild-type strain (SM-01) with a much smaller increase in catalase activity. Microbial pigments, including prodigiosin [43Gulani C, Bhattacharya S, Das A. Assessment of process parameters influencing the enhanced production of prodigiosin from Serratia marcescens and evaluation of its antimicrobial, antioxidant and dyeing potentials. Malays J Microbiol 2012; 8(2): 116-22., 44Vijayalakshmi K, Jagathy K. Production of prodigiosin from Serratia marcescens and its antioxidant and anticancer potential. Int J Adv Res Biolsci 2016; 3(3): 75-88.], provide another potential protective mechanism to oxidative stress such as ozone exposure and have been shown to have antioxidant properties; although the precise mechanism of prodigiosin’s antioxidant properties is unclear, pigments such as prodigiosin act as antioxidants when they donate electrons to reactive oxygen species [45Kim J-S, Han Y-J, Heo M-S. Free radical scavenging activity of Zooshikella sp. JE-34 secondary metabolites by electron spin resonance spectrometry. J Biotechnol 2008; 136(Suppl.): S70.
[http://dx.doi.org/10.1016/j.jbiotec.2008.07.155]
]. Our studies, however, have not found this to be the case. Other protective enzymes are also activated in response to oxidative stress. Alkyl hydroperoxide reductase (ahp) plays a critical role in removing H2O2 in E. coli [46Seaver LC, Imlay JA. Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J Bacteriol 2001; 183(24): 7173-81.
[http://dx.doi.org/10.1128/JB.183.24.7173-7181.2001] [PMID: 11717276]
] but is saturated at low (10-5M) levels, whereas in Pseudomonas, ahp is part of the oxyR regulon and is more critical in detoxifying H2O2 in biofilms [10Panmanee W, Hassett DJ. Differential roles of OxyR-controlled antioxidant enzymes alkyl hydroperoxide reductase (AhpCF) and catalase (KatB) in the protection of Pseudomonas aeruginosa against hydrogen peroxide in biofilm vs. planktonic culture. FEMS Microbiol Lett 2009; 295(2): 238-44.
[http://dx.doi.org/10.1111/j.1574-6968.2009.01605.x] [PMID: 19456869]
]. Superoxide dismutase (SOD), meanwhile, converts O2- into the somewhat less toxic H2O2. Genes for sodA, sodB and sodC can be found in the genome of Serratia marcescens [34Chung WC, Chen LL, Lo WS, Kuo PA, Tu J, Kuo CH. Complete genome sequence of Serratia marcescens WW4. Genome Announc 2013; 1(2): e0012613.
[http://dx.doi.org/10.1128/genomeA.00126-13] [PMID: 23558532]
] and at least one form of SOD has been isolated [47Maejima K, Miyata K, Tomoda K. A manganese superoxide dismutase from Serratia marcescens. Agric Biol Chem 1983; 47(7): 1537-43.]. However, the role of these enzymes in Serratia’s oxidative stress response has been studied little and remains to be elucidated. Likewise, it remains to be determined if any of these play a role in protecting Serratia from ozone, studies which are now beginning in our lab.

CONCLUSION

Although Serratia, when cultured at pigment-producing temperatures, showed increased survival to ozone and peroxide exposure than when cultured and assayed at higher temperatures, subsequent use of non-pigmented mutants showed no difference in ozone survival, catalase activity or hydrogen peroxide resistance. Neither does it appear that oxyR plays a role in protecting Serratia from ozone exposure, as oxyR mutants exhibit similar ozone survival as wild type cells. It is therefore likely that protective mechanisms other than prodigiosin or catalase, possibly regulated by temperature shift or quorum sensing mechanisms [48Hassett DJ, Ma JF, Elkins JG, et al. Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide. Mol Microbiol 1999; 34(5): 1082-93.
[http://dx.doi.org/10.1046/j.1365-2958.1999.01672.x] [PMID: 10594832]
], contribute to Serratia’s resistance to both ozone and peroxide.

CONFLICT OF INTEREST

The author confirms that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

I wish to thank Dr. Robert Shanks (University of Pittsburgh) for providing many of the Serratia strains used in this study. Funding was provided through Plattsburgh State University of New York’s Departmental of Biological Sciences, College of Arts & Sciences, and a Professional Development grant (PDIAP).

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Endorsements



"Open access will revolutionize 21st century knowledge work and accelerate the diffusion of ideas and evidence that support just in time learning and the evolution of thinking in a number of disciplines."


Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


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