Raffaella ScottiI; Laura NicoliniI; Annarita StringaroII; Roberta GabbianelliI
IServizio Biologico per la Gestione della Sperimentazione Animale, Istituto Superiore di Sanità, Rome, Italy
IIDipartimento di Tecnologia e Salute, Istituto Superiore di Sanità, Rome, Italy
Introduction. Escherichia coli O157:H7 possesses one chromosomal and two prophagic sodC genes encoding for Cu,Zn superoxide dismutases. We evaluated the contribution of sodC genes in biofilm formation and its resistance to hydrogen peroxide.
Methods. The biofilm of sodC deletion mutants has been studied, in presence or absence of hydrogen peroxide, by crystal violet in 96-well plates and Scanning Electron Microscopy on glass coverslips.
Results. Deletion of prophagic sodC genes had no effect on biofilm construction, in contrast to the chromosomal gene deletion. Hydrogen peroxide treatment showed higher cell mortality and morphological alterations in sodC deletion mutants respect to wild type. These effects were related to the biofilm development stage.
Conclusion. The role of the three SodCs is not redundant in biofilm formation and the resistance to oxidative damage. The stage of biofilm development is a crucial factor for an effective sanitization.
Key words: biofilm, oxidative stress, hydrogen peroxide, scanning electron microscopy, superoxide dismutase
A biofilm is a structured community of bacterial cells enclosed in a self-producing exopolysaccharide matrix able to adhere to a biotic or abiotic surface. Biofilm plays a crucial role in the pathogenesis of many chronic human infections, and it has been estimated that more than 60% of all microbial infections are associated with biofilm presence .
Escherichia coli O157:H7 is a human pathogenic bacterium responsible for hemorrhagic colitis and the Hemolytic-Uremic Syndrome. The Center for Disease Control and Prevention reported 1994 cases of illness caused by E. coli O157:H7 in USA in the past 4 years, resulting in 782 cases of hospitalization (www.cdc.gov/foodnet/data/trends/tables/2013/table2a-b.html#table-2a).
E. coli O157:H7 shows the ability to form biofilm on a variety of surfaces. Biofilm allows a broad spread of disease caused by this organism, due to its higher resistance to external stresses, sanitizers and antibiotics . Knowledge of factors that can affect biofilm formation may be useful to control persistent infections [3, 4]. E. coli O157:H7, like the most of enteric pathogens, may undergo oxidative stress caused by reactive oxygen species derived from aerobic metabolism, environmental sources and host immune response [5, 6]. Antioxidant enzymes, as peroxidases and superoxide dismutases, mediate the resistance to reactive oxygen species. Proteomic studies on bacterial communities show that proteins involved in oxidative stress are highly expressed in natural microbial biofilms [7-9].
E. coli O157:H7 possesses three sodC genes encoding for Cu,Zn superoxide dismutase (SodC): one identical to the gene present in the non-pathogenic K12 strain (chromosomal sodC), and the other located in sequences (sodC-F1 and sodC-F2) derived from lambdoid prophages. In a previous work, we demonstrated that the proteins of prophagic origin have different structural/functional features with respect to the enzyme encoded by the chromosomal sodC copy .
The purpose of this study was to compare the ability of sodC mutants and the wild type strains to form biofilm and to resist to hydrogen peroxide (H2O2). In particular, we used the RG101 strain lacking the chromosomal sodC gene, the RG104 strain lacking both the prophagic sodC genes and RG105 strain lacking all three sodC genes.
MATERIALS AND METHODS
Strains and growth conditions
E. coli O157:H7 strains used in this work originated from a clinical isolate ED597 identical to EDL 933 reference strain ATCC 43895 for the sodC gene sequences .
We analyzed the ability of E. coli O157:H7 to form biofilm in modified M9 minimal medium, hereafter named modM9 (0.6% w/v of Na2HPO4, 0.3% w/v of KH2PO4, 0.1% w/v of NH4Cl, 0.5% w/v casamino acid, 0.1% w/v MgSO4 and 0.2% w/v glucose). The cells were grown at 28 ºC for 48 h without shaking in polypropylene 96-well plates (Greiner) or on glass coverslips degreased with acetone before using.
In all experiments we employed a bacterial inoculum of 106 cells ml-1 . In particular, 200 µl was added to the wells of 96-well plates and 6 ml to contact plates of 5 cm diameter (LP italiana), containing the glass coverslips.
To analyze the ability of wild type and sodC deletion mutants to form biofilm under stressful conditions, we used the Zhang et al. method . After an initial cell growth at 28 ºC for 6 h in modM9, the medium was replaced by fresh modM9 or modM9 media supplemented with 250 µg l-1 H2O2 and incubated for an additional 42 h. The biofilm grown on the coverslips was processed as described in Scanning Electron Microscopy (SEM) section, while the biofilm grown in the 96-well plates was stained with the crystal violet (CV) modified method .
Briefly, biofilm was washed three times with PBS, dried at 37 ºC for 5 min, fixed at 60 ºC for 1 h and stained with 0.1% CV. After 20 min at RT, CV staining was removed. The biofilm was rinsed in tap water for three times and the dye was solubilized in dimethylsulphoxide (DMSO) for 15 min . The optical density was determined at 595 nm by a 96-well plate reader (Biotek Instrument mod. ELX808). Each experiment was carried out in a single 96-well plate where eight wells were used for each strain grown in the presence or absence of H2O2.
Hydrogen peroxide assay
To test the sodC deletion mutants susceptibility to the oxidative stress, we treated biofilms with H2O2. The biofilm was formed at 28 ºC for 48 h in modM9 and, after three washing with PBS, the 96-well plates were incubated at 37 ºC for 1 h with PBS or PBS supplemented with 1.5 mg l-1 H2O2. The challenged cells were washed in PBS and detached by treating with 200 µl of Trypsin-EDTA at 37 ºC for 5 min. The number of viable cells was determined by plating on LB agar. The survival percentage was calculated for each strain by the ratio of the colony-forming unit (CFU) obtained after incubation with H2O2 and the CFU obtained after incubation with PBS. Each experiment was carried out in a single 96-wellplate, where four wells were used for each strain in presence or absence of H2O2.
After coverslips treatment with 250 µg l-1 H2O2 at 37 ºC for 1 h, the effects of H2O2 on the morphology of a 48 h-biofilm were evaluated.
Scanning Electron Microscopy (SEM)
Biofilms formed on coverslips of 12 mm diameter were fixed with 2.5% glutaraldehyde in 0.1 g l-1 sodium cacodylate buffer, pH 7.4 at room temperature for 30 min. The fixed cells were then washed three times with the same buffer and post fixed with 1% osmium tetroxide for three weeks at 4 ºC. These samples were washed twice with cacodylate buffer and then dehydrated using a graded alcohol series. After the passage in 100% ethanol, the samples were critical point-dried in CO2 (CPD 030 Balzers device, Bal-Tec, Balzers) and gold coated by sputtering (SCD 040 Balzers device, Bal-Tec). The samples were examined with a Cambridge Stereoscan 360 scanning electron microscope (Cambridge Instruments, Cambridge, United Kingdom).
Data analysis was performed with Excel software (Microsoft Office Excel 2007). The level of significance was calculated by Student's t test.
Ability to form biofilm in modM9 and in stressful condition
CV staining of bacteria grown in modM9 (Figure 1) showed that wild type, as RG104 mutant, produced more biofilm than the mutants lacking chromosomal sodC gene (RG101 and RG105). Furthermore, we observed no significant difference between RG101 and RG105 in biofilm accumulation.
We investigated whether oxidative stress could affect the ability of sodC mutants to form biofilm. Cells were initially grown for 6 h, to allow their adhesion to the surface, and then exposed to 250 µg l-1 of H2O2 for 42 h. Wild type and RG104 strains under stressful conditions formed a comparable amount of biofilm higher than that produced by RG101 and RG105 mutants. Furthermore, H2O2 stimulated biofilm formation in the wild type and RG104 strains, whereas in RG101 and RG105 mutants (Figure 1) we observed no significant effect.
Survival to oxidative stress induced by hydrogen peroxide
Figure 2 shows the different resistance to the oxidative stress of wild type or sodC deletion mutant cells organized in biofilm. The treatment of 96-well plates with 1.5 mg l-1 H2O2 for 1 h caused in all mutants the recovery of a smaller number of cells than in wild type. However, we obtained no statistical difference in survival between the mutants, in agreement with our previous results obtained in planktonic culture grown in LB medium .
Biofilm observation by SEM
In order to determine whether morphological changes occurred in biofilm architecture challenged with H2O2, the biofilm formed on coverslips was analyzed by SEM. We observed no significant difference between wild type and deletion mutants in the organization of 48 h-biofilm obtained in modM9. The SEM micrographs (Figure 3a, 3b) showed a well-organized biofilm consisting of cells surrounded by exopolysaccharide matrix; several cellular pillars and curli filaments attached the cells to the substratum, promoting the formation of an interconnecting mesh between cells (data not shown for RG104 and RG105). After an initial growth of 6 h in modM9, changes in biofilm organization were visible when cells were grown for further 42 h in the presence of 250 µg l-1 of H2O2 (Figure 3c, 3d). A slight cell elongation was the only alteration observed in the wild type (Figure 3c), an effect related to H2O2 mediated injury. The RG104 mutant showed a morphology very similar to the wild type, conserving a densely packed structure (data not shown). On the contrary, the chromosomal sodC deletion mutants exhibited more consistent morphological alterations with a few areas with partial fusion of cellular walls (Figure 3d represents RG101, data not shown for RG105). Morphological changes appeared also in the wild type when 48 h-biofilms were challenged with 250 µg l-1 of H2O2 for 1 h. In fact, the Figure 3e shows extensive areas where the cell-cell network was destroyed, and the cells have lost their structural integrity. However, the biofilm of all deletion mutants showed more severe damages when exposed to H2O2 (Figure 3f represents RG101, data not shown for RG104 and RG105). In this case, the entire biofilm organization was dramatically altered showing a loss of individuality of single cells and a total absence of interconnections.
In a previous research, we compared the functional, structural and regulatory properties of the three SodC enzymes. We demonstrated that, despite the SodCs are all involved in cellular protection against H2O2, the sodC genes located in prophagic sequences are differently regulated with respect to the chromosomal sodC copy and encoded for proteins with distinct structural/functional features . Kim et al.  demonstrated the role of chromosomal sodC gene product (SodC) in E. coli O157:H7 biofilm formation, but they did not consider the prophagic genes in their study. Therefore, in this work we investigated for the first time the possibility that the prophagic sodC genes products (SodC-F1/SodC-F2) may be involved in biofilm formation. Moreover, we studied the role that SodCs play in protecting bacteria, embedded in a biofilm structure, from oxidative stress. Our results indicate the involvement of the only SodC in biofilm formation, excluding any role of prophagic proteins in this process. In fact, the biofilm produced in modM9 by the wild type and the RG104 mutant was higher than that produced by the RG101 and RG105 mutants. In addition, the CV values of single mutant and triple mutant were comparable, the latter appearing to be unaffected by the further absence of prophagic copies. Unexpectedly, the SEM observations (Figure 3a, 3b) did not identify morphological differences between the strains grown in modM9. This result suggested that the SodC does not participate in the biofilm morphology, but it is probably involved in the process of the adhesion to the surface, resulting in higher biofilm production. On the other hand, these differences in cell adhesion ability cannot be fully appreciated by SEM because it is a qualitative and not quantitative investigation method.
Several reports showed that the defence mechanisms against oxidative stress might be involved in biofilm formation [9, 14]. Recent works have shown that in E. coli O157:H7 [8, 14] H2O2 induced sodC genes and that SodCs contributed to the resistance against exogenous reactive oxygen species. In this work we investigated whether the three SodCs are differently involved in the protection against H2O2 of biofilm cells. Our results confirm that E. coli O157: H7 biofilm cells are more resistant to H2O2 than cells in planktonic culture . Furthermore, the wild-type strain showed a higher survival capacity towards H2O2, compared to deletion mutants, that have not showed significant differences, indicating the absence of any additive effect of the deletions. The lower resistance of the sodC deletion mutants to stressful conditions was also observed by SEM analyses, carried out on 48 h-biofilm after 1 h of H2O2 treatment. Although in the wild type biofilm the cellular structures appeared injured, all mutants reacted similarly to the oxidative stress, showing more severe damages. Morphological differences between the deletion mutants appeared only when the H2O2 exposure was continuous. In fact, after 42 h of oxidative treatment RG104 biofilm was structurally comparable to wild type and did not show the same cellular alterations observed in RG101 and RG105 biofilms, suggesting the importance of SodC in the biofilm formation in the presence as well as in the absence of H2O2. This suggestion is reinforced by CV results, which showed a significant increase in H2O2 induced biofilm only in the strains that possess chromosomal sodC copy.
Interestingly, the physiological state of cells at the moment of the insult resulted to be a critical factor for H2O2 resistance. In fact, we obtained different results by exposing to H2O2 for 42 h the developing biofilm (after only 6 h of growth in modM9) or by exposing to H2O2 for 1h the 48 h-biofilm. All strains were able to adapt to the presence of H2O2 for 42 h and to form a biofilm, despite damages observed by SEM (Figure 3c, 3d). Probably, during the initial formation of biofilm certain cells, called persisters , were in a physiological state that allowed them to resist to the oxidizing agent and to proliferate until the maturation of biofilm itself. On the other hand, after 1 h of treatment, survival results and SEM observations (Figure 3e, 3f) revealed that H2O2 damaged all structural organization of the 48 h-biofilm. Therefore, our data indicate that the dose can be lethal or sublethal depending on specific development stage of the biofilm during the application of the biocide. Indeed, we demonstrated that the dose of 250 µg l-1 H2O2 used on a developing biofilm had a sublethal effect, inducing further a biofilm increase. On the contrary, when we used the same dose on a 48 h-biofilm it had a lethal effect destroying the cellular structure. It's known that the use of sublethal doses of bactericidal agents or a prolonged exposure to them could select resistant bacteria  able to tolerate a further dose of the same or other bactericides, thus developing a cross-resistance to several classes of biocides [14, 17]. Therefore, treatment with a biocide needs to be immediately effective to avoid the opposite effect as a further biofilm increase and the onset of some form of resistance.
Taken together, our data exclude that the prophagic sodC genes, unlike the chromosomal sodC, are necessary for an efficient biofilm formation, confirming that the three E. coli SodCs play distinct physiological roles. However, despite their different involvement in biofilm formation, all SodCs contribute to the bacteria protection against the toxic action of exogenous H2O2, when they are organized in biofilm. Finally, this study underlines that the biofilm development phase is critical in order to ensure effective sanitation and to reduce the environmental persistence of pathogens.
We thank Corrado Volpe and Lamberto Camilli for their contribution in preparing the figures. Special thanks to Andrea Battistoni for his fundamental contribution in drafting the manuscript, for scientific support and for constant encouragement.
Conflict of interest statement
There are no potential conflicts of interest or any financial or personal relationships with other people or organizations that could inappropriately bias conduct and findings of this study.
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Address for correspondence:
Servizio Biologico per la Gestione della Sperimentazione Animale, Istituto Superiore di Sanità,
Viale Regina Elena 299, Rome, Italy.
Received on 27 May 2014.
Accepted on 10 February 2015.