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Dispersal of human and plant pathogens biofilms via nitric oxide donors at 4°C

Dispersal of human and plant pathogens biofilms via nitric oxide donors at 4°C Recent studies suggest that nitric oxide donors capable of manipulating nitric oxide-mediated signaling in bacteria could induce dispersal of biofilms. Encased in extracellular polymeric substances, human and plant pathogens within biofilms are significantly more resistant to sanitizers. This is particularly a problem in refrigerated environments where food is processed. In an exercise aimed to study the potential of nitric oxide donors as biofilm dispersal in refrigerated conditions, we compared the ability of different nitric oxide donors (SNAP, NO-aspirin and Noc-5) to dislodge biofilms formed by foodborne, human and plant pathogens treated at 4 °C. The donors SNAP and Noc-5 were efficient in dis- persing biofilms formed by Salmonella enterica, pathogenic Escherichia coli and Listeria innocua. The biomasses were decreased up to 30 % when compared with the untreated controls. When the plant pathogens Pectobacterium sp. and Xanthomonas sp. were tested the dispersion was mainly limited to Pectobacterium carotovorum biofilms, decreas- ing up to 15 % after exposure to molsidomine. Finally, the association of selected nitric oxide donors with sanitizers (DiQuat, H O , peracetic acid and PhenoTek II) was effective in dispersing biofilms. The best dispersal was achieved 2 2 by pre-treating P. carotovorum with molsidomine and then peracetic acid. The synergistic effect was estimated up to ~35 % in dispersal when compared with peracetic acid alone. The association of nitric oxide donors with sanitizers could provide a foundation for an improved sanitization procedure for cleaning refrigerate environments. Keywords: Salmonella enterica, Biofilms, Nitric oxide donors, MAHMA NONOate, Biofilm dispersal, Sanitization release nitric oxide in different ways: pH-dependent Introduction manner, via enzymatic reactions, photo or temperature Nitric oxide has recently attracted attention due to its sensitive manner (Maragos et al. 1991; Keefer et al. 1996; potentiality as signaling molecule and for several biotech- Wang et al. 2005). nological applications (Moncada et al. 1991; Gasco et al. In bacteria, nitric oxide seems to have a dual effect: 1996; Chen et  al. 2013). Nitric oxide is currently used it reduces bacterial adhesion (Charville et  al. 2008) and in medicine mediating vasodilation, and it has recently promotes biofilm dispersal (Barraud et al. 2009a, b; Mar- showed a great potential as a molecule able to dislodge vasi et al. 2014, 2015). Pioneering studies by Barraud and biofilms (Wang et  al. 2005; Barraud et  al. 2006). Dur- co-workers (2006) firstly showed the potential disper - ing biofilm dispersal, nitric oxide works as a messenger sion of biofilm preformed by Pseudomonas aeruginosa. rather than a generic poison (Barraud et al. 2006; Barraud Dispersal was induced with low, sub-lethal concentra- et  al. 2009a, b). It can be delivered as a gas or via donor tions (25–500  nmol/L) of the nitric oxide donor sodium molecules (Wang et  al. 2005; Barraud et  al. 2009b) and nitroprusside (SNP) (Barraud et  al. 2006). Other studies the nitric oxide releasing rate is mediated by the chemical showed the dispersal potential of donors such as molsi- structure of the donor itself (Wang et  al. 2005). Donors domine, MAHMA NONOate, diethylamine NONO- ate diethylammonium, PROLI NONOate (Marvasi et  al. 2014; Barnes et al. 2015). The mechanisms leading to the *Correspondence: m.marvasi@mdx.ac.uk Department of Natural Sciences, School of Science and Technology, NO donor-mediated dispersal of biofilm are not com - Middlesex University, The Burroughs, London NW4 4BT, UK pletely clear, but it appears to function in the transition Full list of author information is available at the end of the article © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Marvasi et al. AMB Expr (2016) 6:49 Page 2 of 9 of sessile biofilm organisms to free-swimming bacteria Materials and methods (Barraud et  al. 2015). Genetic studies have revealed that Bacterial strains and culture media genes involved in nitric oxide signaling are regulated in The pathogenic E. coli strains were isolated from out - both Gram-positive and Gram-negative bacteria showing breaks attributed to vegetables: E. coli O157:H7 LJH0537, a universal regulation of nitric oxide in bacteria (Firoved E. coli O157:H7 LJH1186, E. coli O157:H7 LJH643, E. coli et  al. 2004; Xiong and Liu 2010; Narayanasamy 2011; O145 RM12333 (Selma et  al. 2008). Salmonella enterica Hong et  al. 2014). Biofilms can form recalcitrant reser - (isolated from vegetables outbreaks): S. enterica serovar voirs of bacteria that affect water quality, leading to dis - Typhimurium ATCC14028, sv. Braenderup 04E01347, eases and post-harvest losses. It is clear that an effective Braenderup 04E01556, Braenderup 04E00783, sv. Monte- dispersal and removal of these biofilms can benefit the video LJH519, sv. Javiana ATCC BAA-1593 and sv. New- food industry. port C6.3 (Noel et al. 2010). Listeria innocua ATCC33090 Microbes within biofilms are encased in various poly - was purchased from ATCC (Teddington, Middlesex, mers and are significantly more resistant to sanitizers UK). We were also interested in testing the effect of (Corcoran et  al. 2014). The association of nitric oxide nitric oxide donors on dispersing biofilm formed by plant donor(s) with sanitizers or detergents treatments was pathogens; It is well know that they can form biofilm in suggested as a hurdle technology to improve the effec - irrigation pipes (Narayanasamy 2011; Hong et  al. 2014). tiveness of sanitization (Barraud et al. 2006). The disper - The following plant pathogens were used: Pectobacterium sal of bacteria with nitric oxide donors coupled with the carotovorum SR38, and Xanthomonas oryzae pv.oryzae sanitizers treatment could have a synergistic effect: While J18. All strains were maintained as frozen glycerol nitric oxide induces the transition from biofilm to plank - stocks, and were sub-cultured into Luria–Bertani (Fisher, tonic state, the sanitizer can easily kill free-swimming Waltham, MA, USA), Nutrient Agar (Oxoid, Basingstoke cells. Only limited literature is available on the associa- UK) or Brain Heart Infusion broth (Oxoid, Basingstoke tion of sanitizers and donors. Synergistic effects of H O , UK) media. 2 2 the industrial sanitizer SaniDate 12.0 and the cellulose hydrogel nanocrystal (CNC) in dispersing P. aeruginosa, Nitric oxide donors Salmonella and Escherichia coli were reported at 22  °C. The following criteria were used to select candidate nitric The synergistic effect of 500  nM sodium nitroprusside oxide donors: (1) low/moderate toxicity; (2) no more with 1 ppm H O was very effective; Log 2.5 CFU/cm of than 0.1 % of probable, possible or confirmed human car - 2 2 reduction of P. aeruginosa of CFU recovered from treated cinogenicity according to the International Agency for surfaces was measured. In the other two cases, the asso- Research on Cancer (IARC); (3) low/moderate cost; (4) ciation of SaniDate 12.0 with 10  nM molsidomine and commercial availability. MAHMA NONOate increase the dispersal of Salmonella Nitric oxide donors used in this study: S-nitroso- biofilms by 20 % when compared with the sanitizer alone. N-acetyl-d ,l-penicillamine (SNAP) (Cayman Chemicals, With reference to the synergistic effect of CNC with Ann Arbor, MN, USA), 3-(aminopropyl)-1-hydroxy- 1 mM MAHAMA NONOate, the association of the two 3-isopropyl-2-oxo-1-triazene (Noc-5), 2-(acetyloxy)ben- molecules was able to disperse 1  week-old Salmonella zoic acid 4-(nitroxymethyl)phenyl ester (NO-aspirin), biofilm, otherwise impossible with the sole use of the 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl- donor (Barraud et al. 2009b; Marvasi et al. 2014, 2015). 1-hexanamine (MAHMA NONOate), and molsidomine The effectiveness of nitric oxide donor has been mainly (all from Sigma-Aldrich, St. Louis, MO, USA). For each studied at room temperature (about 22  °C) and only compound, 1 mM stock solutions were prepared in phos- minor evidences show biofilm dispersal at 4 °C (Marvasi phate-buffered saline, pH 7.3 (PBS, Fisher, Waltham, MA, et al. 2014). The advantage to sanitizers cold rooms with - USA) and aliquots were stored at −80 °C. For the assays, out to shot down the system is evident: It saves money, serial dilutions were always prepared fresh in PBS just time and it is the preferential approach in large cold before the experiments and used within 5  min of their walk-in environments. preparation. Biofilm dispersion potential of the mol - Our aim is to measure to what extent the efficacy of ecules was tested on polystyrene and polypropylene 96 selected nitric oxide donors can be used in refrigerated well-plates (Fisher, Waltham, MA, USA). conditions in association with sanitizers. The impli - cations of this observation for industrial applications Biofilm formation and dispersal on plastics are interesting: The ability of the nitric oxide donors to Overnight cultures (10   CFU/mL) grown in appropri- disperse biofilms at 4  °C makes them good candidates ate media were diluted in 1:100 of the following media: for cleaning refrigerated surfaces, common in the food in colony-forming antigen (CFA) (Teplitski et  al. 2006) industry. broth medium for Salmonella and E. coli, Nutrient Agar Marvasi et al. AMB Expr (2016) 6:49 Page 3 of 9 for Pectobacterium carotovorum SR38 (bacterial soft rot), Three biological and four technical replicates for each and Xanthomonas campestris J18 (bacterial spot). For experiment were tested. L. innocua Brain Heart Infusion broth with 1  % glucose (Fisher, Waltham, MA, USA) was used (Marvasi et  al. qPCR to verify the expression of nitric oxide related genes 2014). Hundred microlitre of the diluted cultures were in Salmonella aliquoted into wells of 96-well polypropylene or poly- Five millilitre of planktonic cells exposed at 22 °C to 1 nM styrene plates (Fisher, Waltham, MA, USA). Plates with donor MAHMA NONOate for 45  min or PBS (as con- bacteria were incubated for 18 h at 37 °C for Salmonella, trol) were recollected. Total RNA was extracted from E. coli, L. innocua and 48  h at 30  °C for P. carotovorum samples using mirVana miRNA Isolation Kit (Life SR38, X. oryzae pv.oryzae J18 inside a Ziploc bag to pre- Technologies) according to the manufacturers’ instruc- vent evaporation. Biofilms were formed in the dark in tions. RNA integrity was visualized on 1.3  % agarose gel static incubation. Upon completion of the incubation, electrophoresis. Samples were quantified with Nanodrop medium was removed by aspiration and 200 µL aliquots Spectrophotometer (ThermoFisher Scientific) according of serial dilutions of nitric oxide donors in PBS were to manufacturers’ instructions. DNA was removed with added to the wells with biofilms. Dispersal experiments TURBO DNA-freeTM Kit (Life Technologies). cDNA were conducted at 4  °C for 6  h. Dispersal was measured synthesis was performed by using Transcriptor First by staining the remaining biofilms with 1  % (w/v) crys - Strand cDNA Synthesis Kit (Roche) according with the tal violet in ethanol and de-staining with acetic acid 33 % user manual by using random hexamer primers. qPCR (v/v), as described previously (O’Toole and Kolter 1998; was performed on a qPCR LightCycler 96 System (Life Merritt et  al. 2005). Three biological and four technical Technologies) instrument by using PCRBIO SyGreen Mix replicates for each experiment were tested. Percentage of Hi-ROX (PCR Biosystems). Negative control was carried dispersal was calculated by dividing the optical density of out by using PCR grade water instead of cDNA template. the treated by the control optical density. The result was DNA-free RNA was tested via standard PCR amplifica - multiplied by hundred. When cocktails strains were used, tion to ensure complete removal of genomic DNA prior 10  cell/mL from each strain were mixed in the same pro- cDNA generation by using 16S primers (Marvasi et  al. portion before biofilm formation. 2009). Salmonella genes ygaD, mltB, srlB, and gutQ were tested as genes involved in nitric oxide signaling (Ge et al. Additive effect of the sanitizers with nitric oxide donors 2010), whereas rpoD gene was used as an internal refer- Biofilms of P. carotovorum SR38, S. enterica sv Typhimu - ence gene. qPCR was performed by using the following rium ATCC14028, and L. innocua were set up as above cycles: initial denaturation at 95  °C for 2  min, 40 cycles using overnight cultures of the pathogen diluted 1:100 in of denaturation at 95  °C, annealing at 60 °C and extend- the CFA or Nutrient Agar medium, where appropriate, in ing at 65  °C for 30  s. Primers used in PCR reactions are wells of 96-well polypropylene plates (Fisher, Waltham, shown in Additional file  1. Minimum requirement tests MA, USA). Plates with bacteria were incubated as above to ensure specific amplifications were performed as rec - inside a Ziploc bag. Upon completion of the incubation, ommended by the MIQE Guideline (Bustin et  al. 2009). the medium with planktonic bacteria was removed by PCR amplification efficiency was established by means aspiration and 200 µL aliquots of serial dilutions of nitric of calibration curves. Three biological replicas and two −ΔΔCt oxide donors in PBS were added to the biofilms. As con - technical replicas were done for each gene. Livak (2 ) trol, PBS alone was used. Plates were incubated at 4  °C method was used to analyse genes expression. for 6  h. Upon completion of the incubation, planktonic cells were removed by aspiration, wells were washed with Statistical analysis PBS and 200 µL of the following sanitizers, diluted as per The statistical software JMP (SAS) package was used to manufacturer’s recommendations, were loaded into the perform the one-way ANOVA analysis (p < 0.05). Tukey wells: H O (final concentration 2  % v/v), peracetic acid means separation analysis was performed in order to 2 2 (10 % v/v) (Sigma-Aldrich, St. Louis, MO, USA), quater- group the means. nary ammonium compound Diquat (500 mg/L) (Nufarm, Morrisville, NC, USA) or Pheno-Tek II (0.3 % w/v) (Bio- Results Tek Industries, Atlanta, GA, USA). The biofilms were Biofilm dispersal on polypropylene and polystyrene at 4 °C incubated with sanitizers for 10 min at 4 °C, after which Biofilm dispersal was initially tested on polypropylene biofilm dispersal was measured by staining the remaining (Fig.  1). Exposure to SNAP was particularly effective in biofilms with 1  % crystal violet in ethanol, as described dispersing pathogenic Salmonella, E. coli and L. innocua previously (O’Toole and Kolter 1998; Merritt et al. 2005). biofilms which were dispersed up to 25 % when compared Marvasi et al. AMB Expr (2016) 6:49 Page 4 of 9 Fig. 1 Dispersal of different preformed biofilms by the nitric oxide donors SNAP, Noc-5 and NO-aspirin on polypropylene during exposure at 4 °C. Salmonella and E. coli cocktails: see “Materials and methods” section for details about the strains. Concentrations of the nitric oxide donor are on the x-axis. Residual biofilms were quantified by staining with crystal violet. Error bars are standard errors. Asterisk (*) represents significant different mean when compared with the PBS treatment only (p = 0.05) with the control (Fig. 1a–c). Interestingly, in the dispersal The treatment with NO-aspirin was not efficient as of E. coli we observed an inverse dose-dependent effect, SNAP and Noc-5. Only the pathogenic E. coli cocktail already seen in our previous studies but with different was significantly dispersed up to 20  % when compared donors (Marvasi et al. 2014). with the control (Fig. 1g–i). When biofilms were exposed to Noc-5 the dispersal When biofilms were pre-formed on polystyrene (Fig.  2), was similar as those obtained with SNAP (Fig. 1d–f ). Bio- significant dispersal was measured. SNAP treatments mass of E. coli cocktail, L. innocua and Salmonella cock- were effective for E. coli, Listeria and Salmonella cock- tail were significantly reduced. In particular L. innocua tail, with a dispersal ranging between 15 and 20  % in all biofilm was reduced up to 50 % when compared with the treatments (Fig. 2a–c). The treatment with Noc-5 showed control treated with PBS only (Fig. 1e). significant dispersal on all the strains tested (Fig.  2d–f ). Marvasi et al. AMB Expr (2016) 6:49 Page 5 of 9 Fig. 2 Dispersal of different preformed biofilms by the nitric oxide donors SNAP, Noc-5 and NO-aspirin on polystyrene during exposure at 4 °C. Con- centrations of the nitric oxide donor are on the x-axis. Residual biofilms were quantified by staining with crystal violet. Error bars are standard errors. Asterisk (*) represents significant different mean when compared with the PBS treatment only (p = 0.05) The best dispersal occurred for both L. innocua and Sal - previously identified as a donor with a good dispersal monella, where significant biofilms reduction up to 30 % potential (Marvasi et  al. 2014) and NO-aspirin because was measured when compared with the control. a potential safe molecule for application in agriculture. On polystyrene, NO-aspirin was able to disperse pre- P. carotovorum SR38 biofilms were dispersed up to 10 formed pathogenic E. coli cocktail biofilm up to 20 % with and 30  % in polystyrene and polypropylene, respectively effective concentrations of 10  nM and 10  pM (Fig.  2g). (Fig. 3a, b). NO-aspirin showed only a minor but signifi - Similarly, biofilms formed by Listeria innocua and Sal- cant dispersal on X. oryzae, up to 10  % when compared monella cocktail biofilms were dispersed by ~15 % when with the untreated control (Fig. 3d). compared with the control (Fig. 2h, i). Synergistic effect of different sanitizers with nitric oxide Eec ff t of molsidomine and NO‑aspirin in dispersing donors biofilms formed by plant pathogens For the synergistic experiments we tested the donors Pectobacterium carotovorum SR38 and Xanthomonas with best price/dispersal performance from the cur- oryzae pv.oryzae J18 biofilms were formed on polypro - rent and previous screenings (Marvasi et al. 2014, 2015). pylene and tested with molsidomine and NO-aspirin After revision of potential candidates we chose to test at 4  °C (Fig.  3). Molsidomine has been chosen because Noc-5 from the current screening, while molsidomine Marvasi et al. AMB Expr (2016) 6:49 Page 6 of 9 Fig. 3 Dispersal of different preformed plant pathogens biofilms by molsidomine and NO-aspirin at 4 °C. Concentrations of the nitric oxide donors are on the x-axis. Residual biofilms were quantified by staining with crystal violet. Error bars are standard errors. Asterisk (*) represents significant dif- ferent mean when compared with the PBS treatment only (p = 0.05) and MAHAMA NONOate were retrieved from previous confirm the fine-tuning that the donor MAHMA NON - experiments (Marvasi et  al. 2014, 2015). The association Oate acts on the planktonic cells. To confirm the activa - of sanitizers with nitric oxide donors was tested on plant tion of the nitric oxide metabolic cascade upon exposure and human pathogens in order to measure to what extent of MAHMA NONOate in Salmonella, relative expres- synergistic effects occurred. Listeria. innocua, S. enterica sion of ygaD, mltB, srlR, and gutQ genes, previously iden- and P. carotovorum biofilms were pre-treated with differ - tified as involved to nitric oxide signaling, was measured ent nitric oxide donors for 6 h at 4 °C. Biofilms were then at 22 °C (Ge et al. 2010). All the genes tested were higher exposed to different sanitizers (Pheno-Tek II, peracetic expressed in Salmonella cells upon exposure to 1  nM of acid 10 %, H O 2 %, and Diquat) for 10 min (Fig. 4). Bio- the donor MAHMA NONOate when compared with the 2 2 film formed by L. innocua treated with Noc-5  + H O control. Results showed that all the genes were ~1 log 2 2 2 showed a biofilm reduction up to 10  % when compared more expressed than not treated cells: ygaD 1.68 ±  0.10, with H O treatment alone (Fig. 4a). Significant dispersal mltB 1.61 ± 0.10, srlR 1.03 ± 0.30 and gutQ 0.96 ± 0.10. 2 2 was obtained with S. enterica 14028 biofilms treated with the following combinations: Noc-5  +  H O , MAHMA Discussion 2 2 NONOate  +  peracetic acid and MAHMA NONO- In this study we focused on the effect of off-the-shelf ate  +  PhenoTek II (Fig.  4b–d) showing a dispersal up nitric oxide donors to disperse preformed biofilms at to 10  % less biomass when compared with the sanitizer 37  °C and successively exposed to different donors for alone. Finally, P. carotovorum biofilms dispersal was 6  h at 4  °C, a temperature typically used in refrigerated limited but significant when using the algicide Diquat facilities. (widely used in agriculture) or peracetic acid (Fig. 4e, f ). The screenings presented in this work showed that the dispersals at 4  °C were moderate when compared MAHMA NONOate activates the expression of Salmonella with similar screenings carried out at higher tempera- genes involved in the nitric oxide‑mediated signaling tures between 22 and 25 °C (Barraud et al. 2006; Barraud We were also interested in detecting changes in Salmo- et  al. 2009b; Marvasi et  al. 2015). The comparison with nella gene expression during exposure to nitric oxide to recent literature is difficult since different donors were (See figure on next page.) Fig. 4 Additive effect of different sanitizers in association with nitric oxide donors. MAHMA NONOate: 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)- N-methyl-1-hexanamine; Noc-5: 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene; NO-aspirin: 2-(acetyloxy)benzoic acid 4-(nitroxymethyl) phenyl ester. Bars represent the standard error. Asterisk (*) represents the significant synergistic effect of the nitric oxide donor in association with the sanitizer compared with the sanitizer only Marvasi et al. AMB Expr (2016) 6:49 Page 7 of 9 Marvasi et al. AMB Expr (2016) 6:49 Page 8 of 9 used, however a generalized reduction of the dispersal 5-phosphate isomerase; and srlR—glucitol operon was expected. It is well known that low temperatures repressor. Interestingly, relative expression of Salmonella may slow the nitric oxide releasing rate ultimately affect - mltB, ygaD, gutQ and srlR also increased upon infection ing the dispersal (Wang et  al. 2005). However, beside of macrophages with Salmonella (Ge et  al. 2010). Sus- such moderate dispersal we see potential applications in tained production of nitric oxide endows macrophages industry at low temperature. For example, in continu- with cytostatic or cytotoxic activity against bacteria ous flow water systems the constant application of nitric (MacMicking et al. 1997). According with this result, we oxide donors could control biofilm formation on surfaces speculate that data from recent literature indicate that inaccessible for hand cleaning. such genes may play a central role in nitric oxide detoxifi - It is well known that biofilms are significantly more cation, survival and replication of Salmonella upon expo- resistant to chlorine and other sanitizers (Corcoran et al. sure to nitric oxide. 2014). In this study we have shown that SNAP, Noc-5 Enrichment of sanitizers with nitric oxide donors could and NO-aspirin were effective in reducing 18-h old bio - improve produce safety by expanding the tool-kit of pro- films at 4 °C (Figs.  1, 2, 3). In addition, the association of active practices for GAPs, HACCP and cleaning-in-place selected donors with sanitizers significantly reduced bio - (CIP) protocols. However, before its application further films biomass in a synergistic manner (Fig.  4). Significant studies must be done to: (i) Test the effectiveness of this results are reported for the plant pathogen P. carotovo- combined products on actual industrial environment rum, as well as for Salmonella and L. innocua. Of great which may have multiple pathogens and very strong bio- interest is the dispersal of P. carotovorum with the algi- films; (ii) To identify methods to control the nitric oxide cide Diquat  +  MAHMA NONOate. We can speculate release; (iii) To assess the neutralization/toxicity of the that constant application of such combination could be donors once depleted by the nitric oxide. effectively used in agriculture for cleaning greenhouses or irrigation pipes. Additional file Studies from other authors carried out at room tem- perature measured similar synergistic effects but wider Additional file 1. Primers used in this study. in magnitude: A further ~80 % reduction of surface cov- erage of P. aeruginosa biofilm was measured after the association of 500  nM of sodium nitroprusside (SNP) to Authors’ contributions MMarvasi conceived experiments. MMarvasi, THA wrote the manuscript. 10 mM H O (Barraud et al. 2006, 2009b). When Salmo- 2 2 MMarvasi, THA, RCP performed the statistical analysis. IAD, AS, MMatusze- nella biofilms where treated with MAHMA NONOate wska carried out the experiments. All authors read and approved the final or molsidomine in association with the sanitizer Sani- manuscript. Date 12.0, biomass was reduced of an additional 20  % Author details when compared with SaniDate 12.0 alone (Marvasi et al. 1 Department of Natural Sciences, School of Science and Technology, Mid- 2014). Interestingly, the synergistic effect is not only lim - dlesex University, The Burroughs, London NW4 4BT, UK. Soil and Water Science Department, University of Florida, Gainesville, FL, USA. Department ited to sanitizers but also to antibiotics and detergents. of Microbiology and Mycology, Institute of Biomedical Sciences (ICBM), Uni- The exposure of sodium nitroprusside (500  nM) to P. versity of Chile, Santiago, Chile. aeruginosa greatly enhanced the efficacy of tobramycin, Acknowledgements tetracycline and sodium dodecyl sulfate in the removal We acknowledge Dr. Max Teplitski for the constructive advices about the of established P. aeruginosa biofilms from a glass sur - experiments and the manuscript. face (Barraud et  al. 2006, 2009b). Synergistic effect was Competing interests also identified in the field of the chemistry of hydrogels. The authors declare that they have no competing interests. Encapsulation of MAHMA NONOate and molsidomine within a hydrogel composed of cellulose nanocrystals Ethical approval This article does not contain any studies with human participants or animals has shown a synergistic effect in dispersing Salmonella performed by any of the authors. 1-week old biofilms (Marvasi et al. 2015). Finally, exposure to MAHMA NONOate led to the Funding information This research was supported by funding provided by the Florida Tomato Com- expression of Salmonella ygaD, mltB, srlR, and gutQ mittee, Grant #106486 (MM, University of Florida), by the UC-Davis Center for genes included in the recA-hydN genomic region puta- Produce Safety (MM), Grant #2014-308 (University of Florida) and MM start-up tively involved in nitric oxide-mediated signaling (Mar- Grant from Middlesex University, London. vasi et al. 2014). mltB encodes for membrane-bound lytic Received: 24 May 2016 Accepted: 14 July 2016 murein transglycosylase B; ygaD for a ribonucleoside- diphosphate reductase 2 subunit β; gutQ for an arabinose Marvasi et al. AMB Expr (2016) 6:49 Page 9 of 9 References MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu Barnes RJ, Low JH, Bandi RR, Tay M, Chua F, Aung T, Fane AG, Kjelleberg S, Rice Rev Immunol. 1997;15:323–50. SA. Nitric oxide treatment for the control of reverse osmosis membrane Maragos CM, Morley D, Wink DA, Dunams TM, Saavedra JE, Hoffman A, Bove biofouling. Appl Environ Microbiol. 2015;81:2515–24. doi:10.1128/ AA, Isaac L, Hrabie JA, Keefer LK. Complexes of NO with nucleophiles as AEM.03404-14. agents for the controlled biological release of nitric oxide: vasorelaxant Barraud N, Hassett DJ, Hwang SH, Rice SA, Kjelleberg S, Webb JS. Involvement effects. J Med Chem. 1991;34:3242–7. doi:10.1021/jm00115a013. of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa. J Bacteriol. Marvasi M, Chen C, Carrazana M, Durie IA, Teplitski M. Systematic analysis 2006;188:7344–53. doi:10.1128/JB.00779-06. of the ability of nitric oxide donors to dislodge biofilms formed by Barraud N, Schleheck D, Klebensberger J, Webb JS, Hassett DJ, Rice SA, Salmonella enterica and Escherichia coli O157:H7. AMB Express. 2014;4:42. Kjelleberg S. Nitric oxide signaling in Pseudomonas aeruginosa biofilms doi:10.1186/s13568-014-0042-y. mediates phosphodiesterase activity, decreased cyclic di-GMP levels, Marvasi M, Durie IA, McLamore ES, Vanegas DC, Chaturvedi P. Salmonella enter- and enhanced dispersal. J Bacteriol. 2009a;191:7333–42. doi:10.1128/ ica biofilm-mediated dispersal by nitric oxide donors in association with JB.00975-09. cellulose nanocrystal hydrogels. AMB Express. 2015;5:23. doi:10.1186/ Barraud N, Storey MV, Moore ZP, Webb JS, Rice SA, Kjelleberg S. Nitric oxide- s13568-015-0114-7. mediated dispersal in single- and multi-species biofilms of clinically and Marvasi M, Mastromei G, Vedovato E, Balsamo C, Perito B. Bacterial community industrially relevant microorganisms. Microb Biotechnol. 2009b;2:370–8. analysis on the medieval stained glass window “Natività” in the Florence doi:10.1111/j.1751-7915.2009.00098.x. Cathedral. J Cult Herit. 2009;10:124–33. doi:10.1016/j.culher.2008.08.010. Barraud N, Kelso MJ, Scott AR, Kjelleberg S. Nitric oxide: a key mediator of Merritt JH, Kadouri DE, O’Toole GA. Growing and analyzing static biofilms. biofilm dispersal with applications in infectious diseases. Curr Pharm Des. Curr Protoc Microbiol. 2005;Chapter 1:1B–1. doi:10.1002/9780471729259. 2015;21:31–42. doi:10.2174/1381612820666140905112822. mc01b01s00. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Muel- Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, ler R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The and pharmacology. Pharmacol Rev. 1991;43:109–42. MIQE guidelines: minimum information for publication of quantitative Narayanasamy P. Bacterial and phytoplasmal pathogens. In: Microbial plant real-time PCR experiments. Clin Chem. 2009;55(4):611–22. doi:10.1373/ pathogens-detection and disease diagnosis. Berlin: Springer; 2011. clinchem.2008.112797. Noel JT, Arrach N, Alagely A, McClelland M, Teplitski M. Specific responses Charville GW, Hetrick EM, Geer CB, Schoenfisch MH. Reduced bacterial adhe - of Salmonella enterica to tomato varieties and fruit ripeness identified sion to fibrinogen-coated substrates via nitric oxide release. Biomaterials. by in vivo expression technology. PLoS One. 2010. doi:10.1371/journal. 2008;29:4039–44. doi:10.1016/j.biomaterials.2008.07.005. pone.0012406. Chen M, Yu Q, Sun H. Novel strategies for the prevention and treatment of O’Toole GA, Kolter R. Initiation of biofilm formation in Pseudomonas biofilm related infections. Int J Mol Sci. 2013;14:18488–501. doi:10.3390/ fluorescens WCS365 proceeds via multiple, convergent signal- ijms140918488. ling pathways: a genetic analysis. Mol Microbiol. 1998;28:449–61. Corcoran M, Morris D, De Lappe N, O’Connor J, Lalor P, Dockery P, Cormican doi:10.1046/j.1365-2958.1998.00797.x. M. Commonly used disinfectants fail to eradicate Salmonella enterica Selma MV, Ibáñez AM, Allende A, Cantwell M, Suslow T. Eec ff t of gaseous biofilms from food contact surface materials. Appl Environ Microbiol. ozone and hot water on microbial and sensory quality of cantaloupe and 2014;80:1507–14. doi:10.1128/AEM.03109-13. potential transference of Escherichia coli O157:H7 during cutting. Food Firoved AM, Wood SR, Ornatowski W, Deretic V, Timmins GS. Microarray Microbiol. 2008;25:162–8. doi:10.1016/j.fm.2007.06.003. analysis and functional characterization of the nitrosative stress response Teplitski M, Al-Agely A, Ahmer BMM. Contribution of the SirA regulon to bio- in nonmucoid and mucoid Pseudomonas aeruginosa. J Bacteriol. film formation in Salmonella enterica serovar Typhimurium. Microbiology. 2004;186:4046–50. doi:10.1128/JB.186.12.4046-4050.2004. 2006;152:3411–24. doi:10.1099/mic.0.29118-0. Gasco A, Fruttero R, Sorba G. No-donors: an emerging class of compounds in Wang PG, Cai TB, Taniguchi N, editors. Nitric Oxide Donors: for Pharmaceutical medicinal chemistry. Farmaco. 1996;51:617–35. and Biological Applications. Weinheim: Wiley-VCH Verlag GmbH & Co. Ge S, Danino V, He Q, Hinton JCD, Granfors K. Microarray analysis of KGaA; 2005. doi:10.1002/3527603751.fmatter. response of Salmonella during infection of HLA-B27- transfected Xiong Y, Liu Y. Biological control of microbial attachment: a promising alterna- human macrophage-like U937 cells. BMC Genom. 2010;11:1–13. tive for mitigating membrane biofouling. Appl Microbiol Biotechnol. doi:10.1186/1471-2164-11-456. 2010;86:825–37. doi:10.1007/s00253-010-2463-0. Hong CX, Moorman GW, Wohanka W, Büttner C. Biology, detection and management of plant pathology in irrigation water. St. Paul: American Phytopathological Society; 2014. Keefer LK, Nims RW, Davies KM, Wink DA. “NONOates” (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors: convenient nitric oxide dosage forms. Methods Enzymol. 1996;268:281–93. doi:10.1016/ S0076-6879(96)68030-6. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AMB Express Springer Journals

Dispersal of human and plant pathogens biofilms via nitric oxide donors at 4°C

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Copyright © 2016 by The Author(s)
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
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10.1186/s13568-016-0220-1
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Abstract

Recent studies suggest that nitric oxide donors capable of manipulating nitric oxide-mediated signaling in bacteria could induce dispersal of biofilms. Encased in extracellular polymeric substances, human and plant pathogens within biofilms are significantly more resistant to sanitizers. This is particularly a problem in refrigerated environments where food is processed. In an exercise aimed to study the potential of nitric oxide donors as biofilm dispersal in refrigerated conditions, we compared the ability of different nitric oxide donors (SNAP, NO-aspirin and Noc-5) to dislodge biofilms formed by foodborne, human and plant pathogens treated at 4 °C. The donors SNAP and Noc-5 were efficient in dis- persing biofilms formed by Salmonella enterica, pathogenic Escherichia coli and Listeria innocua. The biomasses were decreased up to 30 % when compared with the untreated controls. When the plant pathogens Pectobacterium sp. and Xanthomonas sp. were tested the dispersion was mainly limited to Pectobacterium carotovorum biofilms, decreas- ing up to 15 % after exposure to molsidomine. Finally, the association of selected nitric oxide donors with sanitizers (DiQuat, H O , peracetic acid and PhenoTek II) was effective in dispersing biofilms. The best dispersal was achieved 2 2 by pre-treating P. carotovorum with molsidomine and then peracetic acid. The synergistic effect was estimated up to ~35 % in dispersal when compared with peracetic acid alone. The association of nitric oxide donors with sanitizers could provide a foundation for an improved sanitization procedure for cleaning refrigerate environments. Keywords: Salmonella enterica, Biofilms, Nitric oxide donors, MAHMA NONOate, Biofilm dispersal, Sanitization release nitric oxide in different ways: pH-dependent Introduction manner, via enzymatic reactions, photo or temperature Nitric oxide has recently attracted attention due to its sensitive manner (Maragos et al. 1991; Keefer et al. 1996; potentiality as signaling molecule and for several biotech- Wang et al. 2005). nological applications (Moncada et al. 1991; Gasco et al. In bacteria, nitric oxide seems to have a dual effect: 1996; Chen et  al. 2013). Nitric oxide is currently used it reduces bacterial adhesion (Charville et  al. 2008) and in medicine mediating vasodilation, and it has recently promotes biofilm dispersal (Barraud et al. 2009a, b; Mar- showed a great potential as a molecule able to dislodge vasi et al. 2014, 2015). Pioneering studies by Barraud and biofilms (Wang et  al. 2005; Barraud et  al. 2006). Dur- co-workers (2006) firstly showed the potential disper - ing biofilm dispersal, nitric oxide works as a messenger sion of biofilm preformed by Pseudomonas aeruginosa. rather than a generic poison (Barraud et al. 2006; Barraud Dispersal was induced with low, sub-lethal concentra- et  al. 2009a, b). It can be delivered as a gas or via donor tions (25–500  nmol/L) of the nitric oxide donor sodium molecules (Wang et  al. 2005; Barraud et  al. 2009b) and nitroprusside (SNP) (Barraud et  al. 2006). Other studies the nitric oxide releasing rate is mediated by the chemical showed the dispersal potential of donors such as molsi- structure of the donor itself (Wang et  al. 2005). Donors domine, MAHMA NONOate, diethylamine NONO- ate diethylammonium, PROLI NONOate (Marvasi et  al. 2014; Barnes et al. 2015). The mechanisms leading to the *Correspondence: m.marvasi@mdx.ac.uk Department of Natural Sciences, School of Science and Technology, NO donor-mediated dispersal of biofilm are not com - Middlesex University, The Burroughs, London NW4 4BT, UK pletely clear, but it appears to function in the transition Full list of author information is available at the end of the article © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Marvasi et al. AMB Expr (2016) 6:49 Page 2 of 9 of sessile biofilm organisms to free-swimming bacteria Materials and methods (Barraud et  al. 2015). Genetic studies have revealed that Bacterial strains and culture media genes involved in nitric oxide signaling are regulated in The pathogenic E. coli strains were isolated from out - both Gram-positive and Gram-negative bacteria showing breaks attributed to vegetables: E. coli O157:H7 LJH0537, a universal regulation of nitric oxide in bacteria (Firoved E. coli O157:H7 LJH1186, E. coli O157:H7 LJH643, E. coli et  al. 2004; Xiong and Liu 2010; Narayanasamy 2011; O145 RM12333 (Selma et  al. 2008). Salmonella enterica Hong et  al. 2014). Biofilms can form recalcitrant reser - (isolated from vegetables outbreaks): S. enterica serovar voirs of bacteria that affect water quality, leading to dis - Typhimurium ATCC14028, sv. Braenderup 04E01347, eases and post-harvest losses. It is clear that an effective Braenderup 04E01556, Braenderup 04E00783, sv. Monte- dispersal and removal of these biofilms can benefit the video LJH519, sv. Javiana ATCC BAA-1593 and sv. New- food industry. port C6.3 (Noel et al. 2010). Listeria innocua ATCC33090 Microbes within biofilms are encased in various poly - was purchased from ATCC (Teddington, Middlesex, mers and are significantly more resistant to sanitizers UK). We were also interested in testing the effect of (Corcoran et  al. 2014). The association of nitric oxide nitric oxide donors on dispersing biofilm formed by plant donor(s) with sanitizers or detergents treatments was pathogens; It is well know that they can form biofilm in suggested as a hurdle technology to improve the effec - irrigation pipes (Narayanasamy 2011; Hong et  al. 2014). tiveness of sanitization (Barraud et al. 2006). The disper - The following plant pathogens were used: Pectobacterium sal of bacteria with nitric oxide donors coupled with the carotovorum SR38, and Xanthomonas oryzae pv.oryzae sanitizers treatment could have a synergistic effect: While J18. All strains were maintained as frozen glycerol nitric oxide induces the transition from biofilm to plank - stocks, and were sub-cultured into Luria–Bertani (Fisher, tonic state, the sanitizer can easily kill free-swimming Waltham, MA, USA), Nutrient Agar (Oxoid, Basingstoke cells. Only limited literature is available on the associa- UK) or Brain Heart Infusion broth (Oxoid, Basingstoke tion of sanitizers and donors. Synergistic effects of H O , UK) media. 2 2 the industrial sanitizer SaniDate 12.0 and the cellulose hydrogel nanocrystal (CNC) in dispersing P. aeruginosa, Nitric oxide donors Salmonella and Escherichia coli were reported at 22  °C. The following criteria were used to select candidate nitric The synergistic effect of 500  nM sodium nitroprusside oxide donors: (1) low/moderate toxicity; (2) no more with 1 ppm H O was very effective; Log 2.5 CFU/cm of than 0.1 % of probable, possible or confirmed human car - 2 2 reduction of P. aeruginosa of CFU recovered from treated cinogenicity according to the International Agency for surfaces was measured. In the other two cases, the asso- Research on Cancer (IARC); (3) low/moderate cost; (4) ciation of SaniDate 12.0 with 10  nM molsidomine and commercial availability. MAHMA NONOate increase the dispersal of Salmonella Nitric oxide donors used in this study: S-nitroso- biofilms by 20 % when compared with the sanitizer alone. N-acetyl-d ,l-penicillamine (SNAP) (Cayman Chemicals, With reference to the synergistic effect of CNC with Ann Arbor, MN, USA), 3-(aminopropyl)-1-hydroxy- 1 mM MAHAMA NONOate, the association of the two 3-isopropyl-2-oxo-1-triazene (Noc-5), 2-(acetyloxy)ben- molecules was able to disperse 1  week-old Salmonella zoic acid 4-(nitroxymethyl)phenyl ester (NO-aspirin), biofilm, otherwise impossible with the sole use of the 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl- donor (Barraud et al. 2009b; Marvasi et al. 2014, 2015). 1-hexanamine (MAHMA NONOate), and molsidomine The effectiveness of nitric oxide donor has been mainly (all from Sigma-Aldrich, St. Louis, MO, USA). For each studied at room temperature (about 22  °C) and only compound, 1 mM stock solutions were prepared in phos- minor evidences show biofilm dispersal at 4 °C (Marvasi phate-buffered saline, pH 7.3 (PBS, Fisher, Waltham, MA, et al. 2014). The advantage to sanitizers cold rooms with - USA) and aliquots were stored at −80 °C. For the assays, out to shot down the system is evident: It saves money, serial dilutions were always prepared fresh in PBS just time and it is the preferential approach in large cold before the experiments and used within 5  min of their walk-in environments. preparation. Biofilm dispersion potential of the mol - Our aim is to measure to what extent the efficacy of ecules was tested on polystyrene and polypropylene 96 selected nitric oxide donors can be used in refrigerated well-plates (Fisher, Waltham, MA, USA). conditions in association with sanitizers. The impli - cations of this observation for industrial applications Biofilm formation and dispersal on plastics are interesting: The ability of the nitric oxide donors to Overnight cultures (10   CFU/mL) grown in appropri- disperse biofilms at 4  °C makes them good candidates ate media were diluted in 1:100 of the following media: for cleaning refrigerated surfaces, common in the food in colony-forming antigen (CFA) (Teplitski et  al. 2006) industry. broth medium for Salmonella and E. coli, Nutrient Agar Marvasi et al. AMB Expr (2016) 6:49 Page 3 of 9 for Pectobacterium carotovorum SR38 (bacterial soft rot), Three biological and four technical replicates for each and Xanthomonas campestris J18 (bacterial spot). For experiment were tested. L. innocua Brain Heart Infusion broth with 1  % glucose (Fisher, Waltham, MA, USA) was used (Marvasi et  al. qPCR to verify the expression of nitric oxide related genes 2014). Hundred microlitre of the diluted cultures were in Salmonella aliquoted into wells of 96-well polypropylene or poly- Five millilitre of planktonic cells exposed at 22 °C to 1 nM styrene plates (Fisher, Waltham, MA, USA). Plates with donor MAHMA NONOate for 45  min or PBS (as con- bacteria were incubated for 18 h at 37 °C for Salmonella, trol) were recollected. Total RNA was extracted from E. coli, L. innocua and 48  h at 30  °C for P. carotovorum samples using mirVana miRNA Isolation Kit (Life SR38, X. oryzae pv.oryzae J18 inside a Ziploc bag to pre- Technologies) according to the manufacturers’ instruc- vent evaporation. Biofilms were formed in the dark in tions. RNA integrity was visualized on 1.3  % agarose gel static incubation. Upon completion of the incubation, electrophoresis. Samples were quantified with Nanodrop medium was removed by aspiration and 200 µL aliquots Spectrophotometer (ThermoFisher Scientific) according of serial dilutions of nitric oxide donors in PBS were to manufacturers’ instructions. DNA was removed with added to the wells with biofilms. Dispersal experiments TURBO DNA-freeTM Kit (Life Technologies). cDNA were conducted at 4  °C for 6  h. Dispersal was measured synthesis was performed by using Transcriptor First by staining the remaining biofilms with 1  % (w/v) crys - Strand cDNA Synthesis Kit (Roche) according with the tal violet in ethanol and de-staining with acetic acid 33 % user manual by using random hexamer primers. qPCR (v/v), as described previously (O’Toole and Kolter 1998; was performed on a qPCR LightCycler 96 System (Life Merritt et  al. 2005). Three biological and four technical Technologies) instrument by using PCRBIO SyGreen Mix replicates for each experiment were tested. Percentage of Hi-ROX (PCR Biosystems). Negative control was carried dispersal was calculated by dividing the optical density of out by using PCR grade water instead of cDNA template. the treated by the control optical density. The result was DNA-free RNA was tested via standard PCR amplifica - multiplied by hundred. When cocktails strains were used, tion to ensure complete removal of genomic DNA prior 10  cell/mL from each strain were mixed in the same pro- cDNA generation by using 16S primers (Marvasi et  al. portion before biofilm formation. 2009). Salmonella genes ygaD, mltB, srlB, and gutQ were tested as genes involved in nitric oxide signaling (Ge et al. Additive effect of the sanitizers with nitric oxide donors 2010), whereas rpoD gene was used as an internal refer- Biofilms of P. carotovorum SR38, S. enterica sv Typhimu - ence gene. qPCR was performed by using the following rium ATCC14028, and L. innocua were set up as above cycles: initial denaturation at 95  °C for 2  min, 40 cycles using overnight cultures of the pathogen diluted 1:100 in of denaturation at 95  °C, annealing at 60 °C and extend- the CFA or Nutrient Agar medium, where appropriate, in ing at 65  °C for 30  s. Primers used in PCR reactions are wells of 96-well polypropylene plates (Fisher, Waltham, shown in Additional file  1. Minimum requirement tests MA, USA). Plates with bacteria were incubated as above to ensure specific amplifications were performed as rec - inside a Ziploc bag. Upon completion of the incubation, ommended by the MIQE Guideline (Bustin et  al. 2009). the medium with planktonic bacteria was removed by PCR amplification efficiency was established by means aspiration and 200 µL aliquots of serial dilutions of nitric of calibration curves. Three biological replicas and two −ΔΔCt oxide donors in PBS were added to the biofilms. As con - technical replicas were done for each gene. Livak (2 ) trol, PBS alone was used. Plates were incubated at 4  °C method was used to analyse genes expression. for 6  h. Upon completion of the incubation, planktonic cells were removed by aspiration, wells were washed with Statistical analysis PBS and 200 µL of the following sanitizers, diluted as per The statistical software JMP (SAS) package was used to manufacturer’s recommendations, were loaded into the perform the one-way ANOVA analysis (p < 0.05). Tukey wells: H O (final concentration 2  % v/v), peracetic acid means separation analysis was performed in order to 2 2 (10 % v/v) (Sigma-Aldrich, St. Louis, MO, USA), quater- group the means. nary ammonium compound Diquat (500 mg/L) (Nufarm, Morrisville, NC, USA) or Pheno-Tek II (0.3 % w/v) (Bio- Results Tek Industries, Atlanta, GA, USA). The biofilms were Biofilm dispersal on polypropylene and polystyrene at 4 °C incubated with sanitizers for 10 min at 4 °C, after which Biofilm dispersal was initially tested on polypropylene biofilm dispersal was measured by staining the remaining (Fig.  1). Exposure to SNAP was particularly effective in biofilms with 1  % crystal violet in ethanol, as described dispersing pathogenic Salmonella, E. coli and L. innocua previously (O’Toole and Kolter 1998; Merritt et al. 2005). biofilms which were dispersed up to 25 % when compared Marvasi et al. AMB Expr (2016) 6:49 Page 4 of 9 Fig. 1 Dispersal of different preformed biofilms by the nitric oxide donors SNAP, Noc-5 and NO-aspirin on polypropylene during exposure at 4 °C. Salmonella and E. coli cocktails: see “Materials and methods” section for details about the strains. Concentrations of the nitric oxide donor are on the x-axis. Residual biofilms were quantified by staining with crystal violet. Error bars are standard errors. Asterisk (*) represents significant different mean when compared with the PBS treatment only (p = 0.05) with the control (Fig. 1a–c). Interestingly, in the dispersal The treatment with NO-aspirin was not efficient as of E. coli we observed an inverse dose-dependent effect, SNAP and Noc-5. Only the pathogenic E. coli cocktail already seen in our previous studies but with different was significantly dispersed up to 20  % when compared donors (Marvasi et al. 2014). with the control (Fig. 1g–i). When biofilms were exposed to Noc-5 the dispersal When biofilms were pre-formed on polystyrene (Fig.  2), was similar as those obtained with SNAP (Fig. 1d–f ). Bio- significant dispersal was measured. SNAP treatments mass of E. coli cocktail, L. innocua and Salmonella cock- were effective for E. coli, Listeria and Salmonella cock- tail were significantly reduced. In particular L. innocua tail, with a dispersal ranging between 15 and 20  % in all biofilm was reduced up to 50 % when compared with the treatments (Fig. 2a–c). The treatment with Noc-5 showed control treated with PBS only (Fig. 1e). significant dispersal on all the strains tested (Fig.  2d–f ). Marvasi et al. AMB Expr (2016) 6:49 Page 5 of 9 Fig. 2 Dispersal of different preformed biofilms by the nitric oxide donors SNAP, Noc-5 and NO-aspirin on polystyrene during exposure at 4 °C. Con- centrations of the nitric oxide donor are on the x-axis. Residual biofilms were quantified by staining with crystal violet. Error bars are standard errors. Asterisk (*) represents significant different mean when compared with the PBS treatment only (p = 0.05) The best dispersal occurred for both L. innocua and Sal - previously identified as a donor with a good dispersal monella, where significant biofilms reduction up to 30 % potential (Marvasi et  al. 2014) and NO-aspirin because was measured when compared with the control. a potential safe molecule for application in agriculture. On polystyrene, NO-aspirin was able to disperse pre- P. carotovorum SR38 biofilms were dispersed up to 10 formed pathogenic E. coli cocktail biofilm up to 20 % with and 30  % in polystyrene and polypropylene, respectively effective concentrations of 10  nM and 10  pM (Fig.  2g). (Fig. 3a, b). NO-aspirin showed only a minor but signifi - Similarly, biofilms formed by Listeria innocua and Sal- cant dispersal on X. oryzae, up to 10  % when compared monella cocktail biofilms were dispersed by ~15 % when with the untreated control (Fig. 3d). compared with the control (Fig. 2h, i). Synergistic effect of different sanitizers with nitric oxide Eec ff t of molsidomine and NO‑aspirin in dispersing donors biofilms formed by plant pathogens For the synergistic experiments we tested the donors Pectobacterium carotovorum SR38 and Xanthomonas with best price/dispersal performance from the cur- oryzae pv.oryzae J18 biofilms were formed on polypro - rent and previous screenings (Marvasi et al. 2014, 2015). pylene and tested with molsidomine and NO-aspirin After revision of potential candidates we chose to test at 4  °C (Fig.  3). Molsidomine has been chosen because Noc-5 from the current screening, while molsidomine Marvasi et al. AMB Expr (2016) 6:49 Page 6 of 9 Fig. 3 Dispersal of different preformed plant pathogens biofilms by molsidomine and NO-aspirin at 4 °C. Concentrations of the nitric oxide donors are on the x-axis. Residual biofilms were quantified by staining with crystal violet. Error bars are standard errors. Asterisk (*) represents significant dif- ferent mean when compared with the PBS treatment only (p = 0.05) and MAHAMA NONOate were retrieved from previous confirm the fine-tuning that the donor MAHMA NON - experiments (Marvasi et  al. 2014, 2015). The association Oate acts on the planktonic cells. To confirm the activa - of sanitizers with nitric oxide donors was tested on plant tion of the nitric oxide metabolic cascade upon exposure and human pathogens in order to measure to what extent of MAHMA NONOate in Salmonella, relative expres- synergistic effects occurred. Listeria. innocua, S. enterica sion of ygaD, mltB, srlR, and gutQ genes, previously iden- and P. carotovorum biofilms were pre-treated with differ - tified as involved to nitric oxide signaling, was measured ent nitric oxide donors for 6 h at 4 °C. Biofilms were then at 22 °C (Ge et al. 2010). All the genes tested were higher exposed to different sanitizers (Pheno-Tek II, peracetic expressed in Salmonella cells upon exposure to 1  nM of acid 10 %, H O 2 %, and Diquat) for 10 min (Fig. 4). Bio- the donor MAHMA NONOate when compared with the 2 2 film formed by L. innocua treated with Noc-5  + H O control. Results showed that all the genes were ~1 log 2 2 2 showed a biofilm reduction up to 10  % when compared more expressed than not treated cells: ygaD 1.68 ±  0.10, with H O treatment alone (Fig. 4a). Significant dispersal mltB 1.61 ± 0.10, srlR 1.03 ± 0.30 and gutQ 0.96 ± 0.10. 2 2 was obtained with S. enterica 14028 biofilms treated with the following combinations: Noc-5  +  H O , MAHMA Discussion 2 2 NONOate  +  peracetic acid and MAHMA NONO- In this study we focused on the effect of off-the-shelf ate  +  PhenoTek II (Fig.  4b–d) showing a dispersal up nitric oxide donors to disperse preformed biofilms at to 10  % less biomass when compared with the sanitizer 37  °C and successively exposed to different donors for alone. Finally, P. carotovorum biofilms dispersal was 6  h at 4  °C, a temperature typically used in refrigerated limited but significant when using the algicide Diquat facilities. (widely used in agriculture) or peracetic acid (Fig. 4e, f ). The screenings presented in this work showed that the dispersals at 4  °C were moderate when compared MAHMA NONOate activates the expression of Salmonella with similar screenings carried out at higher tempera- genes involved in the nitric oxide‑mediated signaling tures between 22 and 25 °C (Barraud et al. 2006; Barraud We were also interested in detecting changes in Salmo- et  al. 2009b; Marvasi et  al. 2015). The comparison with nella gene expression during exposure to nitric oxide to recent literature is difficult since different donors were (See figure on next page.) Fig. 4 Additive effect of different sanitizers in association with nitric oxide donors. MAHMA NONOate: 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)- N-methyl-1-hexanamine; Noc-5: 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene; NO-aspirin: 2-(acetyloxy)benzoic acid 4-(nitroxymethyl) phenyl ester. Bars represent the standard error. Asterisk (*) represents the significant synergistic effect of the nitric oxide donor in association with the sanitizer compared with the sanitizer only Marvasi et al. AMB Expr (2016) 6:49 Page 7 of 9 Marvasi et al. AMB Expr (2016) 6:49 Page 8 of 9 used, however a generalized reduction of the dispersal 5-phosphate isomerase; and srlR—glucitol operon was expected. It is well known that low temperatures repressor. Interestingly, relative expression of Salmonella may slow the nitric oxide releasing rate ultimately affect - mltB, ygaD, gutQ and srlR also increased upon infection ing the dispersal (Wang et  al. 2005). However, beside of macrophages with Salmonella (Ge et  al. 2010). Sus- such moderate dispersal we see potential applications in tained production of nitric oxide endows macrophages industry at low temperature. For example, in continu- with cytostatic or cytotoxic activity against bacteria ous flow water systems the constant application of nitric (MacMicking et al. 1997). According with this result, we oxide donors could control biofilm formation on surfaces speculate that data from recent literature indicate that inaccessible for hand cleaning. such genes may play a central role in nitric oxide detoxifi - It is well known that biofilms are significantly more cation, survival and replication of Salmonella upon expo- resistant to chlorine and other sanitizers (Corcoran et al. sure to nitric oxide. 2014). In this study we have shown that SNAP, Noc-5 Enrichment of sanitizers with nitric oxide donors could and NO-aspirin were effective in reducing 18-h old bio - improve produce safety by expanding the tool-kit of pro- films at 4 °C (Figs.  1, 2, 3). In addition, the association of active practices for GAPs, HACCP and cleaning-in-place selected donors with sanitizers significantly reduced bio - (CIP) protocols. However, before its application further films biomass in a synergistic manner (Fig.  4). Significant studies must be done to: (i) Test the effectiveness of this results are reported for the plant pathogen P. carotovo- combined products on actual industrial environment rum, as well as for Salmonella and L. innocua. Of great which may have multiple pathogens and very strong bio- interest is the dispersal of P. carotovorum with the algi- films; (ii) To identify methods to control the nitric oxide cide Diquat  +  MAHMA NONOate. We can speculate release; (iii) To assess the neutralization/toxicity of the that constant application of such combination could be donors once depleted by the nitric oxide. effectively used in agriculture for cleaning greenhouses or irrigation pipes. Additional file Studies from other authors carried out at room tem- perature measured similar synergistic effects but wider Additional file 1. Primers used in this study. in magnitude: A further ~80 % reduction of surface cov- erage of P. aeruginosa biofilm was measured after the association of 500  nM of sodium nitroprusside (SNP) to Authors’ contributions MMarvasi conceived experiments. MMarvasi, THA wrote the manuscript. 10 mM H O (Barraud et al. 2006, 2009b). When Salmo- 2 2 MMarvasi, THA, RCP performed the statistical analysis. IAD, AS, MMatusze- nella biofilms where treated with MAHMA NONOate wska carried out the experiments. All authors read and approved the final or molsidomine in association with the sanitizer Sani- manuscript. Date 12.0, biomass was reduced of an additional 20  % Author details when compared with SaniDate 12.0 alone (Marvasi et al. 1 Department of Natural Sciences, School of Science and Technology, Mid- 2014). Interestingly, the synergistic effect is not only lim - dlesex University, The Burroughs, London NW4 4BT, UK. Soil and Water Science Department, University of Florida, Gainesville, FL, USA. Department ited to sanitizers but also to antibiotics and detergents. of Microbiology and Mycology, Institute of Biomedical Sciences (ICBM), Uni- The exposure of sodium nitroprusside (500  nM) to P. versity of Chile, Santiago, Chile. aeruginosa greatly enhanced the efficacy of tobramycin, Acknowledgements tetracycline and sodium dodecyl sulfate in the removal We acknowledge Dr. Max Teplitski for the constructive advices about the of established P. aeruginosa biofilms from a glass sur - experiments and the manuscript. face (Barraud et  al. 2006, 2009b). Synergistic effect was Competing interests also identified in the field of the chemistry of hydrogels. The authors declare that they have no competing interests. Encapsulation of MAHMA NONOate and molsidomine within a hydrogel composed of cellulose nanocrystals Ethical approval This article does not contain any studies with human participants or animals has shown a synergistic effect in dispersing Salmonella performed by any of the authors. 1-week old biofilms (Marvasi et al. 2015). Finally, exposure to MAHMA NONOate led to the Funding information This research was supported by funding provided by the Florida Tomato Com- expression of Salmonella ygaD, mltB, srlR, and gutQ mittee, Grant #106486 (MM, University of Florida), by the UC-Davis Center for genes included in the recA-hydN genomic region puta- Produce Safety (MM), Grant #2014-308 (University of Florida) and MM start-up tively involved in nitric oxide-mediated signaling (Mar- Grant from Middlesex University, London. vasi et al. 2014). mltB encodes for membrane-bound lytic Received: 24 May 2016 Accepted: 14 July 2016 murein transglycosylase B; ygaD for a ribonucleoside- diphosphate reductase 2 subunit β; gutQ for an arabinose Marvasi et al. AMB Expr (2016) 6:49 Page 9 of 9 References MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu Barnes RJ, Low JH, Bandi RR, Tay M, Chua F, Aung T, Fane AG, Kjelleberg S, Rice Rev Immunol. 1997;15:323–50. SA. Nitric oxide treatment for the control of reverse osmosis membrane Maragos CM, Morley D, Wink DA, Dunams TM, Saavedra JE, Hoffman A, Bove biofouling. Appl Environ Microbiol. 2015;81:2515–24. doi:10.1128/ AA, Isaac L, Hrabie JA, Keefer LK. Complexes of NO with nucleophiles as AEM.03404-14. agents for the controlled biological release of nitric oxide: vasorelaxant Barraud N, Hassett DJ, Hwang SH, Rice SA, Kjelleberg S, Webb JS. Involvement effects. J Med Chem. 1991;34:3242–7. doi:10.1021/jm00115a013. of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa. J Bacteriol. Marvasi M, Chen C, Carrazana M, Durie IA, Teplitski M. Systematic analysis 2006;188:7344–53. doi:10.1128/JB.00779-06. of the ability of nitric oxide donors to dislodge biofilms formed by Barraud N, Schleheck D, Klebensberger J, Webb JS, Hassett DJ, Rice SA, Salmonella enterica and Escherichia coli O157:H7. AMB Express. 2014;4:42. Kjelleberg S. Nitric oxide signaling in Pseudomonas aeruginosa biofilms doi:10.1186/s13568-014-0042-y. mediates phosphodiesterase activity, decreased cyclic di-GMP levels, Marvasi M, Durie IA, McLamore ES, Vanegas DC, Chaturvedi P. Salmonella enter- and enhanced dispersal. J Bacteriol. 2009a;191:7333–42. doi:10.1128/ ica biofilm-mediated dispersal by nitric oxide donors in association with JB.00975-09. cellulose nanocrystal hydrogels. AMB Express. 2015;5:23. doi:10.1186/ Barraud N, Storey MV, Moore ZP, Webb JS, Rice SA, Kjelleberg S. Nitric oxide- s13568-015-0114-7. mediated dispersal in single- and multi-species biofilms of clinically and Marvasi M, Mastromei G, Vedovato E, Balsamo C, Perito B. Bacterial community industrially relevant microorganisms. Microb Biotechnol. 2009b;2:370–8. analysis on the medieval stained glass window “Natività” in the Florence doi:10.1111/j.1751-7915.2009.00098.x. Cathedral. J Cult Herit. 2009;10:124–33. doi:10.1016/j.culher.2008.08.010. Barraud N, Kelso MJ, Scott AR, Kjelleberg S. Nitric oxide: a key mediator of Merritt JH, Kadouri DE, O’Toole GA. Growing and analyzing static biofilms. biofilm dispersal with applications in infectious diseases. Curr Pharm Des. Curr Protoc Microbiol. 2005;Chapter 1:1B–1. doi:10.1002/9780471729259. 2015;21:31–42. doi:10.2174/1381612820666140905112822. mc01b01s00. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Muel- Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, ler R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The and pharmacology. Pharmacol Rev. 1991;43:109–42. MIQE guidelines: minimum information for publication of quantitative Narayanasamy P. Bacterial and phytoplasmal pathogens. In: Microbial plant real-time PCR experiments. Clin Chem. 2009;55(4):611–22. doi:10.1373/ pathogens-detection and disease diagnosis. Berlin: Springer; 2011. clinchem.2008.112797. Noel JT, Arrach N, Alagely A, McClelland M, Teplitski M. Specific responses Charville GW, Hetrick EM, Geer CB, Schoenfisch MH. Reduced bacterial adhe - of Salmonella enterica to tomato varieties and fruit ripeness identified sion to fibrinogen-coated substrates via nitric oxide release. Biomaterials. by in vivo expression technology. PLoS One. 2010. doi:10.1371/journal. 2008;29:4039–44. doi:10.1016/j.biomaterials.2008.07.005. pone.0012406. Chen M, Yu Q, Sun H. Novel strategies for the prevention and treatment of O’Toole GA, Kolter R. Initiation of biofilm formation in Pseudomonas biofilm related infections. Int J Mol Sci. 2013;14:18488–501. doi:10.3390/ fluorescens WCS365 proceeds via multiple, convergent signal- ijms140918488. ling pathways: a genetic analysis. Mol Microbiol. 1998;28:449–61. Corcoran M, Morris D, De Lappe N, O’Connor J, Lalor P, Dockery P, Cormican doi:10.1046/j.1365-2958.1998.00797.x. M. Commonly used disinfectants fail to eradicate Salmonella enterica Selma MV, Ibáñez AM, Allende A, Cantwell M, Suslow T. Eec ff t of gaseous biofilms from food contact surface materials. Appl Environ Microbiol. ozone and hot water on microbial and sensory quality of cantaloupe and 2014;80:1507–14. doi:10.1128/AEM.03109-13. potential transference of Escherichia coli O157:H7 during cutting. Food Firoved AM, Wood SR, Ornatowski W, Deretic V, Timmins GS. Microarray Microbiol. 2008;25:162–8. doi:10.1016/j.fm.2007.06.003. analysis and functional characterization of the nitrosative stress response Teplitski M, Al-Agely A, Ahmer BMM. Contribution of the SirA regulon to bio- in nonmucoid and mucoid Pseudomonas aeruginosa. J Bacteriol. film formation in Salmonella enterica serovar Typhimurium. Microbiology. 2004;186:4046–50. doi:10.1128/JB.186.12.4046-4050.2004. 2006;152:3411–24. doi:10.1099/mic.0.29118-0. Gasco A, Fruttero R, Sorba G. No-donors: an emerging class of compounds in Wang PG, Cai TB, Taniguchi N, editors. Nitric Oxide Donors: for Pharmaceutical medicinal chemistry. Farmaco. 1996;51:617–35. and Biological Applications. Weinheim: Wiley-VCH Verlag GmbH & Co. Ge S, Danino V, He Q, Hinton JCD, Granfors K. Microarray analysis of KGaA; 2005. doi:10.1002/3527603751.fmatter. response of Salmonella during infection of HLA-B27- transfected Xiong Y, Liu Y. Biological control of microbial attachment: a promising alterna- human macrophage-like U937 cells. BMC Genom. 2010;11:1–13. tive for mitigating membrane biofouling. Appl Microbiol Biotechnol. doi:10.1186/1471-2164-11-456. 2010;86:825–37. doi:10.1007/s00253-010-2463-0. Hong CX, Moorman GW, Wohanka W, Büttner C. Biology, detection and management of plant pathology in irrigation water. St. Paul: American Phytopathological Society; 2014. Keefer LK, Nims RW, Davies KM, Wink DA. “NONOates” (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors: convenient nitric oxide dosage forms. Methods Enzymol. 1996;268:281–93. doi:10.1016/ S0076-6879(96)68030-6.

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Published: Jul 26, 2016

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