Microbial Pathogenesis 79 (2015) 8e16
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Different sensitivity levels to norspermidine on biofilm formation in clinical and commensal Staphylococcus epidermidis strains n-Pere z a, Francisco Díaz-Cedillo b, Araceli Contreras-Rodríguez a, Miriam L. Ramo Gabriel Betanzos-Cabrera c, Humberto Peralta d, Sandra Rodríguez-Martínez e, Mario E. Cancino-Diaz e, Janet Jan-Roblero a, Juan C. Cancino Diaz a, * a gicas-Instituto Polit Department of Microbiology, Escuela Nacional de Ciencias Biolo ecnico Nacional, Carpio y Plan de Ayala S/N. Col. Santo Tomas. Deleg. Miguel Hidalgo, C.P. 11340 Mexico City, Mexico b gicas-Instituto Polit Department of Organic Chemistry, Escuela Nacional de Ciencias Biolo ecnico Nacional, Carpio y Plan de Ayala S/N. Col. Santo Tomas. Deleg. Miguel Hidalgo, C.P. 11340 Mexico City, Mexico c noma del Estado de Hidalgo, Mexico Academic Area of Nutrition, Instituto de Ciencias de la Salud, Universidad Auto d micas, Universidad Nacional Auto noma de M Functional Genomics of Prokaryotes Program, Centro de Ciencias Geno exico, Cuernavaca, Mor., Mexico e gicas-Instituto Polit Department of Immunology, Escuela Nacional de Ciencias Biolo ecnico Nacional, Carpio y Plan de Ayala S/N. Col. Santo Tomas. Deleg. Miguel Hidalgo, C.P. 11340 Mexico City, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 September 2014 Received in revised form 22 December 2014 Accepted 26 December 2014 Available online 27 December 2014
Biofilm formation on medical and surgical devices is the main virulence factor of Staphylococcus epidermidis. A recent study has shown that norspermidine inhibits and disassembles the biofilm in the wildtype Bacillus subtilis NCBI3610 strain. In this study, the effect of norspermidine on S. epidermidis biofilm formation of clinical or commensal strains was tested. Biofilm producing strains of S. epidermidis were isolated from healthy skin (HS; n ¼ 3), healthy conjunctiva (HC; n ¼ 9) and ocular infection (OI; n ¼ 19). All strains were treated with different concentrations of norspermidine, spermidine, putrescine, and cadaverine (1, 10, 25, 50 and 100 mM), and the biofilm formation was tested on microtiter plate. Besides, cell-free supernatants of S. epidermidis growth at 4 h and 40 h were analyzed by gas chromatography coupled to mass spectrometry (GCeMS) to detect norspermidine. Results showed that norspermidine at 25 mM and 100 mM prevented the biofilm formation in 45.16% (14/31) and 16.13% (5/31), respectively; only in one isolate from OI, norspermidine did not have effect. Other polyamines as spermidine, putrescine and cadaverine did not have effect on the biofilm formation of the strains tested. Norspermidine was also capable to disassemble a biofilm already formed. Norspermidine was detected in the 40 h cellfree supernatant of S. epidermidis by GCeMS. Norspermidine inhibited the biofilm development of S. epidermidis on the surface of contact lens. In this work, it was demonstrated that S. epidermidis produces and releases norspermidine causing an inhibitory effect on biofilm formation. Moreover, this is the first time showing that clinical S. epidermidis strains have different sensitivity to norspermidine, which suggest that the composition and structure of the biofilms is varied. We propose that norspermidine could potentially be used in the pre-treating of medical and surgical devices to inhibit the biofilm formation. © 2014 Published by Elsevier Ltd.
Keywords: Staphylococcus epidermidis Norspermidine Biofilm Eye
1. Introduction Abbreviations: OI, ocular infection; HC, healthy conjunctiva; HS, healthy skin. n * Corresponding author. Carpio y Plan de Ayala S/N, Col. Santo Tomas, Delegacio Miguel Hidalgo, 11340 Mexico City, Mexico. n-Pere z), [email protected] E-mail addresses: [email protected] (M.L. Ramo (F. Díaz-Cedillo), [email protected] (A. Contreras-Rodríguez), [email protected] (G. Betanzos-Cabrera), [email protected] (H. Peralta), [email protected] (S. Rodríguez-Martínez), mcancinodiaz@gmail. com (M.E. Cancino-Diaz), [email protected] (J. Jan-Roblero), jccancinodiaz@ hotmail.com (J.C. Cancino Diaz). http://dx.doi.org/10.1016/j.micpath.2014.12.004 0882-4010/© 2014 Published by Elsevier Ltd.
Biofilm involves bacterial populations living embedded in a selfproduced extracellular matrix on surfaces and interfaces [1]. Proteins [2] and exopolysaccharides [3] are the major components of the extracellular matrix of the biofilm formed by bacteria, and sometimes also contain extracelular DNA (eDNA) [4]. Biofilms are resistant to antimicrobial agents and to host immune response [5].
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They are the center of many persistent and chronic bacterial infections [5]; such is the case of many device-related infections where the biofilm is critical to cause the infection [6]. In addition, the biofilm is involved in infective endocarditis, urinary tract infections and acute septic arthritis by pathogens such as Staphylococcus aureus and Staphylococcus epidermidis [7,8]. S. epidermidis is recognized as a leading nosocomial pathogen, significantly contributing to the ever-increasing morbidity and mortality of hospital-acquired infections [9]. S. epidermidis plays a major role in biofilm-based medical-device-related infections [10]. Besides, S. epidermidis biofilms in the eyes have been already documented [11,12]. The development of the biofilm is performed in three steps. The first involves the attachment of the cells on biotic or abiotic surfaces. In the second, the cells aggregate and finally, in the third step the mature biofilm is disassembled [13]. Adherence to abiotic surfaces, such as catheters, depends on the hydrophobicity of the surface of bacterial cells [14]. Specific proteins involved in adhesion to abiotic surface in S. epidermidis include autolysin E (AtlE) [15] and biofilm-associated protein (Bap) [16]. While the adherence to biotic surfaces, as occurring in extracellular matrix of the host, is led by the surface proteins called microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), such as SdrG that binds to fibrinogen [17], and SdrF to collagen [18]. After the initial adhesion step, the development of biofilms through intercellular aggregation is mediated by many surface macromolecules. Several S. epidermidis strains secrete poly-N-acetylglucosamine (PNAG), which surrounds the bacterium and enhances biofilm formation [19]. The intercellular adhesion (ica) operon is composed of four genes icaA, icaD, icaB, and icaC, which participate in the biosynthesis of PNAG [20]. Other strains have mutated ica operon and can not produce PNAG, however these strains produce a biofilm constituted of proteins [21]. In the last step of the biofilm's development, the aged biofilm is disrupted or disassembled to form a new biofilm elsewhere. The process of disassembly is not very well understood yet. Recently, Kolodkin-Gal et al. reported that an aged biofilm of Bacillus subtilis NCBI3610 produces D-amino acids [22] and norspermidine [23], and both molecules are involved in the disassembly of the biofilm. They proposed that D-amino acids alter the bond of the TasA protein to the wall cell [22]; however, recently it was demonstrated that the mechanism proposed is not real, and it could be caused by the susceptibility of B. subtilis NCBI3610 to the biofilm-inhibitory effects of D-amino acids during protein synthesis [24]. The norspermidine interacts directly and specifically with the exopolysaccharide causing biofilm's disassembly in B. subtilis NCBI3610 [23]. Norspermidine was able to inhibit biofilm formation in type strains such as S. aureus SC01, B. subtilis NCBI3610 and Escherichia coli MC4100 [23]. However, to date there are no reports about the effect of norspermidine in clinical strains; in addition, the process of disassembly of aged biofilm of S. epidermidis has not been studied. Therefore, the aim of this work was to study the effect of norspermidine on biofilm formation in strains of S. epidermidis from ocular infection. 2. Materials and methods 2.1. Strains Strains used in this study have been previously described [25]. Briefly, three collections of S. epidermidis strains with the ability to form biofilms were isolated from healthy skin (HS; n ¼ 3), healthy conjunctiva (HC; n ¼ 9), and from ocular infections (OI; n ¼ 19). All strains were grown in tryptic-soy broth (TSB; Becton Dickinson, NJ, USA).
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2.2. Biofilm formation determination Semi-quantitative determination of biofilm formation was performed in 96-well tissue culture plates (Nunc, Roskilde, Denmark) based on the method reported by Christensen et al. [26]. An overnight culture was diluted 1:200 with fresh TSB medium and 100 mL were deposited in a 96-well plate and bacteria were grown at 37 C. After 24 h of growth, the plates were washed vigorously with 1X sterile phosphate-buffered saline (PBS), dried for 30 minat 55 C, and stained with 0.5% (w/v) crystal violet solution. After staining, the plates were washed with 1X PBS. A490 nm of the adhered and stained cells was measured using a Multiskan EX Microplate Photometer (Thermo Scientific). According to the criterion proposed by Christensen et al. [26], a strain is considered as nonadherent and therefore biofilm-negative if the value of A490 nm is 0.12; in contrast biofilm forming strains give an A490 nm > 0.12. Besides, Christensen et al. [26] considered a strong biofilm-positive phenotype if the A490 nm was higher than 0.240, and a weak biofilm-positive phenotype if A490 nm was greater than 0.12 but less than or equal to 0.240. Six independent assays were carried out, and the mean of absorbance values was calculated. 2.3. Kinetics of biofilm formation With the aim of determine the kinetics of biofilm formation, an overnight culture was diluted 1:200 with fresh TSB medium and 100 mL were deposited in a removable 96-well plate, with an 12 8 array of well. The plate was incubated at 37 C without agitation and every 4 h one column of 8 well was removed to determined the biofilm formation. It was performed until reach a total of 40 h. To determine if a compound secreted by the bacterium alters the biofilm formation, cell-free supernatants were collected after 4 and 40 h of incubation. Briefly, the preparations of these supernatants were as follow: the bacterium was grown in TSB medium at 37 C for 4 or 40 h without stirring, then the bacterial cultures were centrifuged at 2000 g for 10 min. The supernatants were recovered and filtered through a sterile membrane of 0.22 mm. Biofilm formation was assayed with 100 mL of an overnight culture diluted 1:200 with fresh TSB medium (control), with cell-free supernatants (diluted at 50% in TSB fresh medium), or undiluted supernatants in a 96-well plate. After 24 h of incubation at 37 C without stirring, the culture supernatants were recovered from each well and biofilm formation was determined. The supernatants recovered of each well were assessed at A600 nm, to determine bacterial growth. 2.4. Determination of norspermidine in bacterial supernatants Cell-free supernatants of S. epidermidis obtained at 4 h or 40 h of growth were processed as follows: supernatant (5 mL) was mixed with 2 mL of phosphate-buffered saline solution (pH 7.4; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) and extracted with 5 mL of THF, the organic layer was separated and then dried over anhydrous sodium sulfate. From this solution 1.5 mL was injected to gas chromatography coupled to mass spectrometry (GCeMS) to determinate the content of norspermidine. Norspermidine GCeMS analyses were performed on a Thermo Finnigan capillary gas chromatograph coupled to a mass spectrometer system (model Polaris Q). A fused silica capillary column 5% phenylpoly-dimethyl-siloxane (30 m 0.32 mm, 1.25 mm film thickness) was used under following conditions; oven temperature program from 70 C (5 min) to 200 C at 10 C/min an then to 250 C, the final temperature kept for 5 min; injector temperature 250 C; carrier gas He, flow rate 1 mL/min; the volume of injected sample was 1.5 mL; splitless injection technique; ionization energy 70 eV, in the electronic ionization (EI) mode; ion source temperature
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200 C; scan mass range of m/z 30e700 and interface line temperature 300 C. Norspermidine used as an external standard was dissolved in THF at 10 mg L1 and injected to GCeMS, the retention time was 6.23 min. The identification of the compound was performed comparing its retention time with the external standard. Norspermidine was identified based on comparison of their mass spectra with those gathered in the NIST-MS library.
2.5. Inhibition and disassembly of biofilm by polyamines For the biofilm formation inhibition assay, a volume of 100 mL of 1:200 diluted overnight bacterial culture was added to a 96-well plate with TSB supplemented individually with norspermidine, spermidine, putrescine, or cadaverine at 1, 10, 25, 50 and 100 mM (Sigma, Toluca, Mexico). The plate was incubated at 37 C for 24 h and the biofilm formation was determined as previously described. For the mature biofilm disassembly evaluation, primarily the mature biofilm was allowed to form as follow: a volume of 100 mL of 1:200 diluted overnight bacterial culture was added to a 96-well plate with TSB medium and the plate was incubated at 37 C for 24 h. Later the wells were washed with 1X PBS and norspermidine was incorporated to different concentrations. The plate was incubated for 6 h more and posteriorly biofilm formation was measured as described above.
2.6. Biofilm formation inhibition by norspermidine on the surface of contact lenses Silicone-hydrogel contact lenses containing 42% (v/v) Filcon 1B and 58% (v/v) water were used to evaluate the effect of norspermidine on S. epidermidis biofilm on the surface of contact lenses. The lenses containing 42% (v/v) Filcon 1B and 58% (v/v) water (Acuvue, Johnson & Johnson Vision Care) were washed twice with cold sterile PBS and placed in a 24-well plate, one contact lens per well. Later, 2 mL of TSB or TSB containing 25 mM norspermidine were added to each well; followed by the addition of 100 mL of 1:200 diluted overnight bacterial culture. The plate was incubated at 37 C for 24 h. After that, the contact lenses were washed vigorously with 1X PBS, and stained with Calceine AM (Invitrogen, Carlsbad, CA, USA) for 15 min. The biofilm was observed using a confocal scanning laser microscopy (Zeiss Axiovert 200M) with an argon laser for excitation at 488 nm (green fluorescence). Images were captured and processed by using LSM5 Pascal software.
3. Results 3.1. Formation of S. epidermidis biofilm Biofilm formation was assayed each 4 h for 40 h of incubation in order to determine the period to form a mature biofilm and the disassembly of it. Biofilm formation in S. epidermidis 96 OI strain (isolated from ocular infection) was analyzed and three main events were exhibited: in the first 24 h, the formation of the biofilm took place; in the second, the maturation of the biofilm occurred during the next 4 h; and in the third stage, the disassembly of the mature biofilm had effect in the last 8 h, as it is shown in Fig. 1A. This result showed that after a long incubation (32e40 h), S. epidermidis biofilm was disassembled and suggests that a compound produced by the bacterium was responsible for this event. With the aim of know if the bacterium produces a compound involving in the disassembly of the mature biofilm, biofilm formation of 96 IO strain was assayed using a 40 h cell-free supernatant, and fresh TBS medium (control). Biofilm formation was inhibited significantly (p < 0.05) in the presence of 40 h cell-free supernatant (either diluted or undiluted) compared to the control (Fig. 1B). To discard that the inhibition of biofilm is caused by the low concentration of nutrients present in the 40 h cell-free supernatant, bacterial growth was determined. Measurements at A600nm of the control supernatant and 40 h cell-free supernatants after biofilm formation were similar, discarding the above hypothesis. This result suggests that a component present in the 40 h cell-free supernatant was able to inhibit the biofilm formation in S. epidermidis. 3.2. Detection of norspermidine produced by S. epidermidis According to the results described above and based on recent evidence supporting the disassembler effect of norspermidine on biofilms, GCeMS analyses were performed with 4 h and 40 h cellfree supernatants of S. epidermidis 96 OI strain to identify the disruptive compound. It was detected a peak with a retention time of 6.23 min in the 40 h cell-free supernatant (Fig. 2A) but it was absent in the 4 h cell-free supernatant (Fig. 2C). The detected peak in the 40 h cell-free supernatant coincided with the nospermidine external standard (Fig. 2B), with the same retention time of 6.23 min. The mass spectra for both peaks (40 h cell-free supernatant and external standard) were similar (Fig. 2D and E) and comparing them with those gathered in the NIST-MS library, they corresponded to 3,30 -iminobispropylamine (norspermidine). These
Fig. 1. Aged media has an effect on biofilm formation by S. epidermidis. Biofilm formation was followed for 40 h in S. epidermidis 96 strain obtained from ocular infection (96 OI) as described in the Materials and methods section (A). The effect of 40 h cell-free supernatant on biofilm formation in S. epidermidis 96 OI strain was conducted (B). In both cases, biofilm was measured after incubation with fresh TSB (C, control), a 1:2 mixture of fresh TSB and 40 h cell-free supernatant (50/50%) and non-diluted 40 h cell-free supernatant (100% AM, aged culture medium). The assays were performed in six independent assays and analyzed using a one-way ANOVA with a Tukey's test. Asterisks indicate statistical significance compared with non-treated cells.
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Fig. 2. Norspermidine is present in S. epidermidis culture supernatant. Detection of norspermidine in the 40 h cell-free supernatant of S. epidermidis 96 OI strain by GCeMS. Chromatographs of the 40 h cell-free supernatant of S. epidermidis (A), norspermidine external standard (B), and 4 h cell-free supernatant (C). Mass spectra of the peak (6.23 min) of the 40 h cell-free supernatant of S. epidermidis (D), external standard (E).
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results demonstrated that S. epidermidis produced norspermidine after 40 h. Norspermidine concentration at 40 h cell-free supernatant was 326.7 mM. Therefore it could suggest that norspermidine has a role in the inhibition and disassembly of mature biofilm in S. epidermidis.
3.3. Effect of exogeneous norspermidine in S. epidermidis biofilm formation Based on the results that S. epidermidis produces norspermidine after 40 h, exogenous norspermidine was added to evaluate its effect on biofilm formation in strains from HS and HC (hereon called commensals) and from OI (pathogenic). All strains assayed produced strong biofilm phenotypes (A490 nm was higher than 0.240), except 144 IO strain (Table 1). Inhibition or no inhibition of biofilm formation was the effect caused by nospermidine in all the strains tested. The concentrations of 1 and 50 mM of norspermidine prevented the biofilm formation in 13% of the strains (4/31), with percent inhibition for 1 mM from 72.3 to 85.4%, and for 50 mM from 61.9 to 72.1%. While, the norspermidite at 100 mM inhibited biofilm formation in 16.13% of the strains tested (5/31), with a range of inhibition of 62.4e75%. Norspermidine had the highest inhibitory effect on biofilm formation (45.16%; 14/31) at 25 mM, with a percent inhibition from 52.2 to 83.1% (Table 1). Also biofilm formation of the
control strain S. epidermidis RP62A was inhibited at 25 mM of norspermidine. Interestingly, in one strain (named as 1654 from OI) was not inhibited the biofilm formation by exogeneous norspermidine even at the highest concentration. In general norspermidine had negative effect on biofilm formation in the 30 strains tested (Table 1) and there was not significant difference (P > 0.05) between the three different sources of isolation. With the aim to observe if other polyamines could inhibit the biofilm formation, spermidine, putrescine and cadaverine were assayed at different concentrations. Spermidine, putrescine and cadaverine did not inhibit biofilm formation in the 96 OI strain (Fig. 3A), the same result was observed with the rest of S. epidermidis strains (n ¼ 30). This result clearly showed that norspermidine was the only able to inhibit the biofilm formation and this could be due to its structure. Similarly, in a previous work we demonstrated that D-amino acids could also inhibit the biofilm formation in Staphylococcus epidermidis [27]. We tested the effect of D-phenylalanine (individual and combined with norspermidine) on biofilm formation. The biofilm formation in the 96 OI strain was more inhibited with the mixture of norspermidine and D-phenylalanine than with the individual compounds (Fig. 3B). However, the strain 1654 IO was not inhibited neither by the mixture of norspermidine and D-phenylalanine, nor by individual D-phenylalanine (data not shown).
Table 1 Percent inhibition of norspermidine on biofilm formation in isolates from IO, HC and HS. Source of isolationa IO 96 106 159 105 1655 156 151 155 104 158 214 56 125 68 2038 144 157 64 1654 HC 103 132 152 13 31 52 61 14 107 HS 4 35 37 RP62A
1 mM (%)b
10 mM (%)b
25 mM (%)b
50 mM (%)b
100 mM (%)b
% Disassembly (100 mM)
0.611 0.758 0.575 0.415 0.702 0.687 0.66 0.355 0.801 0.599 0.472 0.398 1.614 1.641 0.15225 0.392 1.757 0.853 1.671
85.4 75.2 14.2 13.1 10.1 1.5 2.7 2 0 0 12.9 13.2 1.9 14.2 8.4 15.1 0 0 0
87.6 76.4 71.1 21.6 21.7 8.6 10.1 1 0.8 12.6 17.1 18.5 8 12 14.2 12.8 1 0 0
89 77.4 73.5 55.4 55.7 62.6 59.9 54.9 53 52.8 52.2 59.3 19.5 15 17 13.9 3 3.8 0
91.7 81.4 75.5 63.7 58.9 65.9 63.8 58.9 62.5 63.5 57.5 61.7 61.9 63.4 20.4 22 6.9 17.7 0
93.2 86.7 79.1 74.7 69.6 68 66.3 59.8 67.4 68 62.7 64.1 75.2 68.6 62.4 75 69.5 66 1.9
57.9 58.8 18.6 2.7 52.1 62.4 57.2 0 10.6 9 64.8 6.6 6.6 17 25.9 2 4.5 7.5 8.6
0.430 0.805 1.759 1.387 0.489 1.389 1.193 1.328 0.430
72.3 76.6 3.2 10.7 0 0 0 0 22.3
78.3 75.8 63.3 8.7 13.6 11.2 8.9 0.6 28.3
81.4 74.6 71.6 82.4 54.6 83.1 83.1 10.6 24.4
82.6 80.4 73.1 84.6 57.2 85.8 84.2 72.1 26.1
82.3 82.4 82.3 90 62.4 87.5 86 71.9 65.5
27.9 70.8 0 11.5 0 1.7 85.9 6.4 11.1
1.103 0.36 0.73 0.521
11.3 0 7.2 10.1
70.1 6.4 22.7 21.6
69.5 59.3 22.24 56.8
73.5 63.7 68.1 62.7
79.2 70.8 67.4 75.7
69.8 12.8 65.7 52.3
Biofilm* (A490
nm)
The percentages in bold indicate significant difference (p < 0.05) between A490 nm of treatment with norspermidine and A490 nm of control* (formation of biofilm without norspermidine). Biofilm formed in the absence of norspermidine and considered as 0% of biofilm inhibition. a OI, ocular infection; HC, healthy conjunctiva; and HS, healthy skin. b Percent inhibition was calculated with reference to measuring at A490 nm of biofilm formed without norspermidine (0% of biofilm inhibition). Biofilm formation inhibition by norspermidine was conducted as described in the Material and methods section and the assays were performed in six independent assays and analyzed with a one-way ANOVA with a Tukey's test. There was not significant difference (P > 0.05) between the three different sources of isolation.
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Fig. 3. Effect of polyamines and D-amino acids on biofilm formation. Biofilm formation was determined using the 96 IO strain in either fresh TSB (C, control) or TSB supplemented with norspermidine (Nors, 100 mM), spermidine (Spe, 100 mM), putrescine (Put, 100 mM), and cadaverine (Cad, 100 mM) (A). Biofilm formation was tested using a mixture of norspermidine (Nors, 100 mM) and D-phenylalanine (Phe, 100 mM) in the 96 IO strain (B). The assays were performed in six independent assays and analyzed using a one-way ANOVA with a Tukey's test. Asterisks indicate statistical significance compared with non-treated cells.
The effect of norspermidine on the growth of S. epidermidis during a kinetic of 8 h was evaluated. The growth of S. epidermidis was not modified by norspermidine, thus the same concentration of cells was obtained in the culture with norspermidine and in the control without norspermidine (Fig. S1). These results showed that norspermidine has an inhibitory effect on the biofilm formation but no effect on the bacterial growth. 3.4. Mature biofilm disassembly by exogeneous norspermidine In order to know if norspermidine could be able to disassemble an already formed and mature biofilm, a mature biofilm of 24 h was washed, added with norspermidine, incubated for 6 h and evaluated at A490 nm. Norspermidine (at 25 or 100 mM) was able to disassemble the mature biofilm in the 96 IO strain (Fig. 4A). Once again, norspermidine had not effect on the 1654 IO strain (Fig. 4B). Disassembly caused by nospermidine took place in 10 strains from a total of 31 (32.25%) at 100 mM (Table 1). The same result was observed with S. epidermidis RP62A strain. 3.5. Norspermidine inhibits S. epidermidis biofilm formation on contact lenses surface For the reason that one of the main means of causing S. epidermidis ocular infection are the contact lenses, the use of norspermidine to prevent the biofilm formation in contact lenses was assayed. Results showed that norspermidine inhibited biofilm formation on the lenses after 24 h of incubation (Fig. 5B) while biofilm was formed on non-treated lens (Fig. 5A). A graphic computational analysis shows the biofilm formation on the lenses non-treated, while the biofilm was not detectable on the lens treated with norspermidine (Fig. 5A and B). This assay was performed with three strains and the same result was obtained. 4. Discussion Polyamines are organic polycations that at physiological pH are positively charged [28]. They are involved in several cell processes, including DNA and RNA function modulation, protection against oxidative degeneration, regulation of bacterial porins and ionchannels in eukaryotic cells [29e33]. Recently, a new function of norspermidine was described acting as a biofilm formation inhibitor in B. subtilis NCBI3610, S. aureus SCO1 and E. coli MC4100 [23].
In this work was demonstrated that S. epidermidis produces and releases norspermidine into the culture medium after 40 h of incubation. Moreover, the addition of exogenous norspermidine inhibits biofilm formation. Nevertheless, the effect of norspermidine on S. epidermidis strains from IO, HS or HC was varied and different sensitivity levels to norspermidine were observed; in addition, biofilm formation in one strain from IO was not inhibited by norspermidine. This finding shows that the effect of norspermidine on biofilm formation depends on the strain and the so-called universal effect of norspemidine that Kolodkin-Gal et al. [23] showed for B. subtilis NCBI3610, S. aureus SCO1 and E. coli MC4100 strains is not accurate. This work contributes to know natural disassembly of a mature biofilm in S. epidermidis. By means of the kinetic study of biofilm formation in S. epidermidis, it was determined that the biofilm reaches the maturity after 24 h of incubation; after which, it began to be disassembled, possibly due to the production of specific compounds. This premise was supported by the inhibition of biofilm formation in young bacterial cell exposed to 40 h cell-free supernatant (Fig. 1B). However, it should not be excluded that the biofilm's disassembly at 24 h could occur due to the decrease in nutrients of the medium because the medium was not replaced. Furthermore, norspermidine was found in the 40 h cell-free supernatant and no traces of norspermidine were detected in 4 h cellfree supernatant of S. epidermidis. In another study, it was demonstrated that S. epidermidis produces D-amino acids in the stationary phase of growth and these D-amino acids also inhibit biofilm formation [27]. Thus, the results presented here suggest that after the formation of a mature biofilm, S. epidermidis produces norspermidine and D-amino acids to trigger the disassembly, and subsequent the release of the bacteria to form a new biofilm elsewhere. When norspermidine was added exogenously, biofilm formation in-vitro was inhibited; nevertheless, varying levels of sensitivity were found in the strains assayed. This suggests that S. epidermidis strains form biofilms of different biochemical composition, or with same biochemical composition but different structural arrangement. Besides, norspermidine bonds specifically to the exopolysaccharides of the biofilm of B. subtilis NCBI3610 strain [23] indicating that the target of norspermidine is exopolysaccharides. The variation in levels of sensitivity to norspermidine of the isolates tested could be explained by the following reasons: i) the different content of exopolysaccharides in the biofilms of the
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Fig. 4. Norspermidine is able to disassemble S. epidermidis biofilms. (A) S. epidermidis 96 IO strain was used to test the ability of norspermidine (Nors, 25 or 100 mM) to disassemble the biofilm. Firstly, it was allowed the biofilm formation for 24 h at 37 C in TSB (development of mature biofilm), after washing with PBS, norspermidine was added. Biofilm was measured after 6 h of incubation. (B) It shows the result for the 1654 IO strain, which was resistant to biofilm disassembly. The assays were performed six times and analyzed using a one-way ANOVA with a Tukey's test. Asterisks indicate statistical significance compared with non-treated cells.
Fig. 5. Norspermidine inhibits S. epidermidis biofilm formation on contact lenses surface. Biofilm formation was determined using either fresh TSB (A) or TSB supplemented with 25 mM norspermidine (B) on a contact lens. Images were acquired by confocal scanning laser microscopy (CSLM, Carl Zeiss Axiovert 200M) using 488 nm wavelength excitation and a 10X objective. Images were captured and processed using LSM5 Pascal software.
strains. In a previous study was demonstrated that most of the strains used in this work produce biofilm type protein- or protein/ eDNA, with a low content of exopolysaccharide [34]. However, in most of the strains, the norspermidine was required at high concentration to inhibit biofilm formation, therefore this interpretation is not viable; ii) the structural arrangement of exopolysaccharide within the biofilm; this might be the best alternative, because in these isolates the proteins or eDNA into their biofilms are the major component, and they might be covering or protecting to the exopolysaccharides toward the action of norspermidine; and finally, iii) the strains also contain eDNA within its biofilms, and norspermidine could be joined to eDNA instead of exopolysaccharides. Consequently, these results suggest that biofilms of S. epidermidis strains are complex and hence the effect of norspermidine on them is heterogenous. Previously, Kolodkin-Gal et al. [23] proposed that norspermidine is also capable to disassemble the mature biofilm of B. subtilis NCBI3610 strain. In the present work, it was found that norspermidine has a similar effect on S. epidermidis' mature biofilms, confirming the proposal by Kolodkin-Gal et al. [23]. However, the disassembly by norspermidine did not take place in all S. epidermidis strains assayed (32.25%; 10/31). To our knowledge there are not studies demonstrating the disassembly on biofilms of S. epidermidis or S. aureus by norspermidine, therefore this is the
first work that highlights this effect. The disassembly mechanism that norspermidine causes is based on its symmetrical structure (i.e. two lateral propylamines, not present in spermidine), which perfectly attaches to the negatively charged sugar residues (e.g. urionic acids), or to neutral sugars with polar groups (e.g. poly-nacetylglucosamine), causing the collapse of the exopolysaccharides and the disassembly of the biofilm [23]. In addition, we propose that norspermidine is not the sole compound acting on the biofilm, due to in some strains did not occur biofilm disassembly by norspermidine. Other compounds working in conjunction with norspermidine could disrupt biofilm's integrity. This hypothesis is based on the results obtained when the mixture of norspermidine and D-phenylalanine was assayed, and it was observed an increased inhibitory effect on biofilm formation. Furthermore, some strains may produce a more robust biofilm, which make them more resistant to the disassembly by norspermidine, and therefore such strains may have a higher level of virulence than sensitive norspermidine strains. Even though norspermidine was not able to disassemble the biofilms of all isolates, this compound may be used to cover medical equipment, in order to prevent the biofilm formation of S. epidermidis, evidenced by the inhibition assays on the surface of contact lens. Recently Hobley et al. [35] published a manuscript, which contradicts the findings reported by Kolodkin-gal et al. [23], wherein
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they exhibited that B. subtilis NCBI3610 does not produce norspermidine due to the lack of the key enzymes (carboxynorspermidine dehydrogenase, and carboxynorspermidine decarboxylase) that synthesize the compound; therefore, norspermidine could not inhibit biofilm formation [35]. We do not have an explication to Hobley's et al. [35] work, however it is worth mentioning that they did not include to Staphylococcus strains; the authors only focused on B. subtilis NCBI3610 strain, therefore their finding should not be extrapolated with the obtained for Staphylococcus strains. The findings reported in this work are similar to those obtained by Kolodkin-gal et al. [23] and support the premise that norspermidine inhibits S. epidermidis biofilm formation. However, we do not exclude that norspermidine could have a different effect to the reported here; such as in Vibrio cholerae, where norspermidine induces biofilm formation due to the effect of the NspS transmembrane protein [36]. Besides, in other work was demonstrated that norspermidine disassembles a specific type of old-aged, large microbial aggregate formed by a mixed culture [37]. Our results show clearly that the action of norspermidine on biofilms is varied and its physiological effects could depend on the bacterium and the strain assayed. In summary, in this work was demonstrated that clinical and commensal strains of S. epidermidis have different sensitivity to inhibition of biofilm formation by norspermidine, and also norspemidine is capable to disassemble a mature biofilm. This is the first study demonstrating different sensitivity levels to norspermidine by clinical and commensal strains of S. epidermidis. We propose that a mixture of norspermidine and D-amino acids may be incorporated into medical devices to prevent biofilm formation by this bacterium. Acknowledgments This work was supported by a grant from “Consejo Nacional de Ciencia y Tecnología (CONACyT) No. 153268”. FDC, ACR, SRM, MECD, JJR, and JCCD appreciate the COFAA and EDI, IPN fellowships; and support from the SNI-CONACyT. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micpath.2014.12.004. References [1] G. O'Toole, H.B. Kaplan, R. Kolter, Biofilm formation as microbial development, Annu Rev. Microbiol. 54 (2000) 49e79, http://dx.doi.org/10.1146/ annurev.micro.54.1.49. [2] G. Kogan, I. Sadovskaya, P. Chaignon, A. Chokr, S. Jabbouri, Biofilms of clinical strains of Staphylococcus that do not contain polysaccharide intercellular adhesin, FEMS Microbiol. Lett. 255 (2006) 11e16, http://dx.doi.org/10.1111/ j.1574-6968.2005.00043.x. [3] J. Wingender, M. Strathmann, A. Rode, A. Leis, H.C. Flemming, Isolation and biochemical characterization of extracellular polymeric substances from Pseudomonas aeruginosa, Methods Enzymol. 336 (2001) 302e314. [4] K.C. Rice, E.E. Mann, J.L. Endres, E.C. Weiss, J.E. Cassat, M.S. Smeltzer, K.W. Bayles, The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus, Proc. Natl. Acad. Sci. U S A 104 (2007) 8113e8118, http://dx.doi.org/10.1073/pnas.0610226104. [5] J.W. Costerton, P.S. Stewart, E.P. Greenberg, Bacterial biofilms: a common cause of persistent infections, Science 284 (1999) 1318e1322, http:// dx.doi.org/10.1126/science.284.5418.1318. [6] P.D. Fey, Modality of bacterial growth presents unique targets: how do we treat biofilm-mediated infections? Curr. Opin. Microbiol. 13 (2010) 610e615, http://dx.doi.org/10.1016/j.mib.2010.09.007. [7] J.D. Bryers, Medical biofilms, Biotechnol. Bioeng. 100 (2008) 1e18, http:// dx.doi.org/10.1002/bit.21838. [8] M. Otto, Staphylococcal biofilms, Curr. Top. Microbiol. Immunol. 322 (2008) 207e228. [9] K.L. Rogers, P.D. Fey, M.E. Rupp, Coagulase-negative staphylococcal infections, Infect. Dis. Clin. North Am. 23 (2009) 73e98, http://dx.doi.org/10.1016/
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Different sensitivity levels to norspermidine on biofilm formation in clinical and commensal Staphylococcus epidermidis strains n-Pere z a, Francisco Díaz-Cedillo b, Araceli Contreras-Rodríguez a, Miriam L. Ramo Gabriel Betanzos-Cabrera c, Humberto Peralta d, Sandra Rodríguez-Martínez e, Mario E. Cancino-Diaz e, Janet Jan-Roblero a, Juan C. Cancino Diaz a, * a gicas-Instituto Polit Department of Microbiology, Escuela Nacional de Ciencias Biolo ecnico Nacional, Carpio y Plan de Ayala S/N. Col. Santo Tomas. Deleg. Miguel Hidalgo, C.P. 11340 Mexico City, Mexico b gicas-Instituto Polit Department of Organic Chemistry, Escuela Nacional de Ciencias Biolo ecnico Nacional, Carpio y Plan de Ayala S/N. Col. Santo Tomas. Deleg. Miguel Hidalgo, C.P. 11340 Mexico City, Mexico c noma del Estado de Hidalgo, Mexico Academic Area of Nutrition, Instituto de Ciencias de la Salud, Universidad Auto d micas, Universidad Nacional Auto noma de M Functional Genomics of Prokaryotes Program, Centro de Ciencias Geno exico, Cuernavaca, Mor., Mexico e gicas-Instituto Polit Department of Immunology, Escuela Nacional de Ciencias Biolo ecnico Nacional, Carpio y Plan de Ayala S/N. Col. Santo Tomas. Deleg. Miguel Hidalgo, C.P. 11340 Mexico City, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 September 2014 Received in revised form 22 December 2014 Accepted 26 December 2014 Available online 27 December 2014
Biofilm formation on medical and surgical devices is the main virulence factor of Staphylococcus epidermidis. A recent study has shown that norspermidine inhibits and disassembles the biofilm in the wildtype Bacillus subtilis NCBI3610 strain. In this study, the effect of norspermidine on S. epidermidis biofilm formation of clinical or commensal strains was tested. Biofilm producing strains of S. epidermidis were isolated from healthy skin (HS; n ¼ 3), healthy conjunctiva (HC; n ¼ 9) and ocular infection (OI; n ¼ 19). All strains were treated with different concentrations of norspermidine, spermidine, putrescine, and cadaverine (1, 10, 25, 50 and 100 mM), and the biofilm formation was tested on microtiter plate. Besides, cell-free supernatants of S. epidermidis growth at 4 h and 40 h were analyzed by gas chromatography coupled to mass spectrometry (GCeMS) to detect norspermidine. Results showed that norspermidine at 25 mM and 100 mM prevented the biofilm formation in 45.16% (14/31) and 16.13% (5/31), respectively; only in one isolate from OI, norspermidine did not have effect. Other polyamines as spermidine, putrescine and cadaverine did not have effect on the biofilm formation of the strains tested. Norspermidine was also capable to disassemble a biofilm already formed. Norspermidine was detected in the 40 h cellfree supernatant of S. epidermidis by GCeMS. Norspermidine inhibited the biofilm development of S. epidermidis on the surface of contact lens. In this work, it was demonstrated that S. epidermidis produces and releases norspermidine causing an inhibitory effect on biofilm formation. Moreover, this is the first time showing that clinical S. epidermidis strains have different sensitivity to norspermidine, which suggest that the composition and structure of the biofilms is varied. We propose that norspermidine could potentially be used in the pre-treating of medical and surgical devices to inhibit the biofilm formation. © 2014 Published by Elsevier Ltd.
Keywords: Staphylococcus epidermidis Norspermidine Biofilm Eye
1. Introduction Abbreviations: OI, ocular infection; HC, healthy conjunctiva; HS, healthy skin. n * Corresponding author. Carpio y Plan de Ayala S/N, Col. Santo Tomas, Delegacio Miguel Hidalgo, 11340 Mexico City, Mexico. n-Pere z), [email protected] E-mail addresses: [email protected] (M.L. Ramo (F. Díaz-Cedillo), [email protected] (A. Contreras-Rodríguez), [email protected] (G. Betanzos-Cabrera), [email protected] (H. Peralta), [email protected] (S. Rodríguez-Martínez), mcancinodiaz@gmail. com (M.E. Cancino-Diaz), [email protected] (J. Jan-Roblero), jccancinodiaz@ hotmail.com (J.C. Cancino Diaz). http://dx.doi.org/10.1016/j.micpath.2014.12.004 0882-4010/© 2014 Published by Elsevier Ltd.
Biofilm involves bacterial populations living embedded in a selfproduced extracellular matrix on surfaces and interfaces [1]. Proteins [2] and exopolysaccharides [3] are the major components of the extracellular matrix of the biofilm formed by bacteria, and sometimes also contain extracelular DNA (eDNA) [4]. Biofilms are resistant to antimicrobial agents and to host immune response [5].
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They are the center of many persistent and chronic bacterial infections [5]; such is the case of many device-related infections where the biofilm is critical to cause the infection [6]. In addition, the biofilm is involved in infective endocarditis, urinary tract infections and acute septic arthritis by pathogens such as Staphylococcus aureus and Staphylococcus epidermidis [7,8]. S. epidermidis is recognized as a leading nosocomial pathogen, significantly contributing to the ever-increasing morbidity and mortality of hospital-acquired infections [9]. S. epidermidis plays a major role in biofilm-based medical-device-related infections [10]. Besides, S. epidermidis biofilms in the eyes have been already documented [11,12]. The development of the biofilm is performed in three steps. The first involves the attachment of the cells on biotic or abiotic surfaces. In the second, the cells aggregate and finally, in the third step the mature biofilm is disassembled [13]. Adherence to abiotic surfaces, such as catheters, depends on the hydrophobicity of the surface of bacterial cells [14]. Specific proteins involved in adhesion to abiotic surface in S. epidermidis include autolysin E (AtlE) [15] and biofilm-associated protein (Bap) [16]. While the adherence to biotic surfaces, as occurring in extracellular matrix of the host, is led by the surface proteins called microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), such as SdrG that binds to fibrinogen [17], and SdrF to collagen [18]. After the initial adhesion step, the development of biofilms through intercellular aggregation is mediated by many surface macromolecules. Several S. epidermidis strains secrete poly-N-acetylglucosamine (PNAG), which surrounds the bacterium and enhances biofilm formation [19]. The intercellular adhesion (ica) operon is composed of four genes icaA, icaD, icaB, and icaC, which participate in the biosynthesis of PNAG [20]. Other strains have mutated ica operon and can not produce PNAG, however these strains produce a biofilm constituted of proteins [21]. In the last step of the biofilm's development, the aged biofilm is disrupted or disassembled to form a new biofilm elsewhere. The process of disassembly is not very well understood yet. Recently, Kolodkin-Gal et al. reported that an aged biofilm of Bacillus subtilis NCBI3610 produces D-amino acids [22] and norspermidine [23], and both molecules are involved in the disassembly of the biofilm. They proposed that D-amino acids alter the bond of the TasA protein to the wall cell [22]; however, recently it was demonstrated that the mechanism proposed is not real, and it could be caused by the susceptibility of B. subtilis NCBI3610 to the biofilm-inhibitory effects of D-amino acids during protein synthesis [24]. The norspermidine interacts directly and specifically with the exopolysaccharide causing biofilm's disassembly in B. subtilis NCBI3610 [23]. Norspermidine was able to inhibit biofilm formation in type strains such as S. aureus SC01, B. subtilis NCBI3610 and Escherichia coli MC4100 [23]. However, to date there are no reports about the effect of norspermidine in clinical strains; in addition, the process of disassembly of aged biofilm of S. epidermidis has not been studied. Therefore, the aim of this work was to study the effect of norspermidine on biofilm formation in strains of S. epidermidis from ocular infection. 2. Materials and methods 2.1. Strains Strains used in this study have been previously described [25]. Briefly, three collections of S. epidermidis strains with the ability to form biofilms were isolated from healthy skin (HS; n ¼ 3), healthy conjunctiva (HC; n ¼ 9), and from ocular infections (OI; n ¼ 19). All strains were grown in tryptic-soy broth (TSB; Becton Dickinson, NJ, USA).
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2.2. Biofilm formation determination Semi-quantitative determination of biofilm formation was performed in 96-well tissue culture plates (Nunc, Roskilde, Denmark) based on the method reported by Christensen et al. [26]. An overnight culture was diluted 1:200 with fresh TSB medium and 100 mL were deposited in a 96-well plate and bacteria were grown at 37 C. After 24 h of growth, the plates were washed vigorously with 1X sterile phosphate-buffered saline (PBS), dried for 30 minat 55 C, and stained with 0.5% (w/v) crystal violet solution. After staining, the plates were washed with 1X PBS. A490 nm of the adhered and stained cells was measured using a Multiskan EX Microplate Photometer (Thermo Scientific). According to the criterion proposed by Christensen et al. [26], a strain is considered as nonadherent and therefore biofilm-negative if the value of A490 nm is 0.12; in contrast biofilm forming strains give an A490 nm > 0.12. Besides, Christensen et al. [26] considered a strong biofilm-positive phenotype if the A490 nm was higher than 0.240, and a weak biofilm-positive phenotype if A490 nm was greater than 0.12 but less than or equal to 0.240. Six independent assays were carried out, and the mean of absorbance values was calculated. 2.3. Kinetics of biofilm formation With the aim of determine the kinetics of biofilm formation, an overnight culture was diluted 1:200 with fresh TSB medium and 100 mL were deposited in a removable 96-well plate, with an 12 8 array of well. The plate was incubated at 37 C without agitation and every 4 h one column of 8 well was removed to determined the biofilm formation. It was performed until reach a total of 40 h. To determine if a compound secreted by the bacterium alters the biofilm formation, cell-free supernatants were collected after 4 and 40 h of incubation. Briefly, the preparations of these supernatants were as follow: the bacterium was grown in TSB medium at 37 C for 4 or 40 h without stirring, then the bacterial cultures were centrifuged at 2000 g for 10 min. The supernatants were recovered and filtered through a sterile membrane of 0.22 mm. Biofilm formation was assayed with 100 mL of an overnight culture diluted 1:200 with fresh TSB medium (control), with cell-free supernatants (diluted at 50% in TSB fresh medium), or undiluted supernatants in a 96-well plate. After 24 h of incubation at 37 C without stirring, the culture supernatants were recovered from each well and biofilm formation was determined. The supernatants recovered of each well were assessed at A600 nm, to determine bacterial growth. 2.4. Determination of norspermidine in bacterial supernatants Cell-free supernatants of S. epidermidis obtained at 4 h or 40 h of growth were processed as follows: supernatant (5 mL) was mixed with 2 mL of phosphate-buffered saline solution (pH 7.4; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) and extracted with 5 mL of THF, the organic layer was separated and then dried over anhydrous sodium sulfate. From this solution 1.5 mL was injected to gas chromatography coupled to mass spectrometry (GCeMS) to determinate the content of norspermidine. Norspermidine GCeMS analyses were performed on a Thermo Finnigan capillary gas chromatograph coupled to a mass spectrometer system (model Polaris Q). A fused silica capillary column 5% phenylpoly-dimethyl-siloxane (30 m 0.32 mm, 1.25 mm film thickness) was used under following conditions; oven temperature program from 70 C (5 min) to 200 C at 10 C/min an then to 250 C, the final temperature kept for 5 min; injector temperature 250 C; carrier gas He, flow rate 1 mL/min; the volume of injected sample was 1.5 mL; splitless injection technique; ionization energy 70 eV, in the electronic ionization (EI) mode; ion source temperature
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200 C; scan mass range of m/z 30e700 and interface line temperature 300 C. Norspermidine used as an external standard was dissolved in THF at 10 mg L1 and injected to GCeMS, the retention time was 6.23 min. The identification of the compound was performed comparing its retention time with the external standard. Norspermidine was identified based on comparison of their mass spectra with those gathered in the NIST-MS library.
2.5. Inhibition and disassembly of biofilm by polyamines For the biofilm formation inhibition assay, a volume of 100 mL of 1:200 diluted overnight bacterial culture was added to a 96-well plate with TSB supplemented individually with norspermidine, spermidine, putrescine, or cadaverine at 1, 10, 25, 50 and 100 mM (Sigma, Toluca, Mexico). The plate was incubated at 37 C for 24 h and the biofilm formation was determined as previously described. For the mature biofilm disassembly evaluation, primarily the mature biofilm was allowed to form as follow: a volume of 100 mL of 1:200 diluted overnight bacterial culture was added to a 96-well plate with TSB medium and the plate was incubated at 37 C for 24 h. Later the wells were washed with 1X PBS and norspermidine was incorporated to different concentrations. The plate was incubated for 6 h more and posteriorly biofilm formation was measured as described above.
2.6. Biofilm formation inhibition by norspermidine on the surface of contact lenses Silicone-hydrogel contact lenses containing 42% (v/v) Filcon 1B and 58% (v/v) water were used to evaluate the effect of norspermidine on S. epidermidis biofilm on the surface of contact lenses. The lenses containing 42% (v/v) Filcon 1B and 58% (v/v) water (Acuvue, Johnson & Johnson Vision Care) were washed twice with cold sterile PBS and placed in a 24-well plate, one contact lens per well. Later, 2 mL of TSB or TSB containing 25 mM norspermidine were added to each well; followed by the addition of 100 mL of 1:200 diluted overnight bacterial culture. The plate was incubated at 37 C for 24 h. After that, the contact lenses were washed vigorously with 1X PBS, and stained with Calceine AM (Invitrogen, Carlsbad, CA, USA) for 15 min. The biofilm was observed using a confocal scanning laser microscopy (Zeiss Axiovert 200M) with an argon laser for excitation at 488 nm (green fluorescence). Images were captured and processed by using LSM5 Pascal software.
3. Results 3.1. Formation of S. epidermidis biofilm Biofilm formation was assayed each 4 h for 40 h of incubation in order to determine the period to form a mature biofilm and the disassembly of it. Biofilm formation in S. epidermidis 96 OI strain (isolated from ocular infection) was analyzed and three main events were exhibited: in the first 24 h, the formation of the biofilm took place; in the second, the maturation of the biofilm occurred during the next 4 h; and in the third stage, the disassembly of the mature biofilm had effect in the last 8 h, as it is shown in Fig. 1A. This result showed that after a long incubation (32e40 h), S. epidermidis biofilm was disassembled and suggests that a compound produced by the bacterium was responsible for this event. With the aim of know if the bacterium produces a compound involving in the disassembly of the mature biofilm, biofilm formation of 96 IO strain was assayed using a 40 h cell-free supernatant, and fresh TBS medium (control). Biofilm formation was inhibited significantly (p < 0.05) in the presence of 40 h cell-free supernatant (either diluted or undiluted) compared to the control (Fig. 1B). To discard that the inhibition of biofilm is caused by the low concentration of nutrients present in the 40 h cell-free supernatant, bacterial growth was determined. Measurements at A600nm of the control supernatant and 40 h cell-free supernatants after biofilm formation were similar, discarding the above hypothesis. This result suggests that a component present in the 40 h cell-free supernatant was able to inhibit the biofilm formation in S. epidermidis. 3.2. Detection of norspermidine produced by S. epidermidis According to the results described above and based on recent evidence supporting the disassembler effect of norspermidine on biofilms, GCeMS analyses were performed with 4 h and 40 h cellfree supernatants of S. epidermidis 96 OI strain to identify the disruptive compound. It was detected a peak with a retention time of 6.23 min in the 40 h cell-free supernatant (Fig. 2A) but it was absent in the 4 h cell-free supernatant (Fig. 2C). The detected peak in the 40 h cell-free supernatant coincided with the nospermidine external standard (Fig. 2B), with the same retention time of 6.23 min. The mass spectra for both peaks (40 h cell-free supernatant and external standard) were similar (Fig. 2D and E) and comparing them with those gathered in the NIST-MS library, they corresponded to 3,30 -iminobispropylamine (norspermidine). These
Fig. 1. Aged media has an effect on biofilm formation by S. epidermidis. Biofilm formation was followed for 40 h in S. epidermidis 96 strain obtained from ocular infection (96 OI) as described in the Materials and methods section (A). The effect of 40 h cell-free supernatant on biofilm formation in S. epidermidis 96 OI strain was conducted (B). In both cases, biofilm was measured after incubation with fresh TSB (C, control), a 1:2 mixture of fresh TSB and 40 h cell-free supernatant (50/50%) and non-diluted 40 h cell-free supernatant (100% AM, aged culture medium). The assays were performed in six independent assays and analyzed using a one-way ANOVA with a Tukey's test. Asterisks indicate statistical significance compared with non-treated cells.
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Fig. 2. Norspermidine is present in S. epidermidis culture supernatant. Detection of norspermidine in the 40 h cell-free supernatant of S. epidermidis 96 OI strain by GCeMS. Chromatographs of the 40 h cell-free supernatant of S. epidermidis (A), norspermidine external standard (B), and 4 h cell-free supernatant (C). Mass spectra of the peak (6.23 min) of the 40 h cell-free supernatant of S. epidermidis (D), external standard (E).
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results demonstrated that S. epidermidis produced norspermidine after 40 h. Norspermidine concentration at 40 h cell-free supernatant was 326.7 mM. Therefore it could suggest that norspermidine has a role in the inhibition and disassembly of mature biofilm in S. epidermidis.
3.3. Effect of exogeneous norspermidine in S. epidermidis biofilm formation Based on the results that S. epidermidis produces norspermidine after 40 h, exogenous norspermidine was added to evaluate its effect on biofilm formation in strains from HS and HC (hereon called commensals) and from OI (pathogenic). All strains assayed produced strong biofilm phenotypes (A490 nm was higher than 0.240), except 144 IO strain (Table 1). Inhibition or no inhibition of biofilm formation was the effect caused by nospermidine in all the strains tested. The concentrations of 1 and 50 mM of norspermidine prevented the biofilm formation in 13% of the strains (4/31), with percent inhibition for 1 mM from 72.3 to 85.4%, and for 50 mM from 61.9 to 72.1%. While, the norspermidite at 100 mM inhibited biofilm formation in 16.13% of the strains tested (5/31), with a range of inhibition of 62.4e75%. Norspermidine had the highest inhibitory effect on biofilm formation (45.16%; 14/31) at 25 mM, with a percent inhibition from 52.2 to 83.1% (Table 1). Also biofilm formation of the
control strain S. epidermidis RP62A was inhibited at 25 mM of norspermidine. Interestingly, in one strain (named as 1654 from OI) was not inhibited the biofilm formation by exogeneous norspermidine even at the highest concentration. In general norspermidine had negative effect on biofilm formation in the 30 strains tested (Table 1) and there was not significant difference (P > 0.05) between the three different sources of isolation. With the aim to observe if other polyamines could inhibit the biofilm formation, spermidine, putrescine and cadaverine were assayed at different concentrations. Spermidine, putrescine and cadaverine did not inhibit biofilm formation in the 96 OI strain (Fig. 3A), the same result was observed with the rest of S. epidermidis strains (n ¼ 30). This result clearly showed that norspermidine was the only able to inhibit the biofilm formation and this could be due to its structure. Similarly, in a previous work we demonstrated that D-amino acids could also inhibit the biofilm formation in Staphylococcus epidermidis [27]. We tested the effect of D-phenylalanine (individual and combined with norspermidine) on biofilm formation. The biofilm formation in the 96 OI strain was more inhibited with the mixture of norspermidine and D-phenylalanine than with the individual compounds (Fig. 3B). However, the strain 1654 IO was not inhibited neither by the mixture of norspermidine and D-phenylalanine, nor by individual D-phenylalanine (data not shown).
Table 1 Percent inhibition of norspermidine on biofilm formation in isolates from IO, HC and HS. Source of isolationa IO 96 106 159 105 1655 156 151 155 104 158 214 56 125 68 2038 144 157 64 1654 HC 103 132 152 13 31 52 61 14 107 HS 4 35 37 RP62A
1 mM (%)b
10 mM (%)b
25 mM (%)b
50 mM (%)b
100 mM (%)b
% Disassembly (100 mM)
0.611 0.758 0.575 0.415 0.702 0.687 0.66 0.355 0.801 0.599 0.472 0.398 1.614 1.641 0.15225 0.392 1.757 0.853 1.671
85.4 75.2 14.2 13.1 10.1 1.5 2.7 2 0 0 12.9 13.2 1.9 14.2 8.4 15.1 0 0 0
87.6 76.4 71.1 21.6 21.7 8.6 10.1 1 0.8 12.6 17.1 18.5 8 12 14.2 12.8 1 0 0
89 77.4 73.5 55.4 55.7 62.6 59.9 54.9 53 52.8 52.2 59.3 19.5 15 17 13.9 3 3.8 0
91.7 81.4 75.5 63.7 58.9 65.9 63.8 58.9 62.5 63.5 57.5 61.7 61.9 63.4 20.4 22 6.9 17.7 0
93.2 86.7 79.1 74.7 69.6 68 66.3 59.8 67.4 68 62.7 64.1 75.2 68.6 62.4 75 69.5 66 1.9
57.9 58.8 18.6 2.7 52.1 62.4 57.2 0 10.6 9 64.8 6.6 6.6 17 25.9 2 4.5 7.5 8.6
0.430 0.805 1.759 1.387 0.489 1.389 1.193 1.328 0.430
72.3 76.6 3.2 10.7 0 0 0 0 22.3
78.3 75.8 63.3 8.7 13.6 11.2 8.9 0.6 28.3
81.4 74.6 71.6 82.4 54.6 83.1 83.1 10.6 24.4
82.6 80.4 73.1 84.6 57.2 85.8 84.2 72.1 26.1
82.3 82.4 82.3 90 62.4 87.5 86 71.9 65.5
27.9 70.8 0 11.5 0 1.7 85.9 6.4 11.1
1.103 0.36 0.73 0.521
11.3 0 7.2 10.1
70.1 6.4 22.7 21.6
69.5 59.3 22.24 56.8
73.5 63.7 68.1 62.7
79.2 70.8 67.4 75.7
69.8 12.8 65.7 52.3
Biofilm* (A490
nm)
The percentages in bold indicate significant difference (p < 0.05) between A490 nm of treatment with norspermidine and A490 nm of control* (formation of biofilm without norspermidine). Biofilm formed in the absence of norspermidine and considered as 0% of biofilm inhibition. a OI, ocular infection; HC, healthy conjunctiva; and HS, healthy skin. b Percent inhibition was calculated with reference to measuring at A490 nm of biofilm formed without norspermidine (0% of biofilm inhibition). Biofilm formation inhibition by norspermidine was conducted as described in the Material and methods section and the assays were performed in six independent assays and analyzed with a one-way ANOVA with a Tukey's test. There was not significant difference (P > 0.05) between the three different sources of isolation.
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Fig. 3. Effect of polyamines and D-amino acids on biofilm formation. Biofilm formation was determined using the 96 IO strain in either fresh TSB (C, control) or TSB supplemented with norspermidine (Nors, 100 mM), spermidine (Spe, 100 mM), putrescine (Put, 100 mM), and cadaverine (Cad, 100 mM) (A). Biofilm formation was tested using a mixture of norspermidine (Nors, 100 mM) and D-phenylalanine (Phe, 100 mM) in the 96 IO strain (B). The assays were performed in six independent assays and analyzed using a one-way ANOVA with a Tukey's test. Asterisks indicate statistical significance compared with non-treated cells.
The effect of norspermidine on the growth of S. epidermidis during a kinetic of 8 h was evaluated. The growth of S. epidermidis was not modified by norspermidine, thus the same concentration of cells was obtained in the culture with norspermidine and in the control without norspermidine (Fig. S1). These results showed that norspermidine has an inhibitory effect on the biofilm formation but no effect on the bacterial growth. 3.4. Mature biofilm disassembly by exogeneous norspermidine In order to know if norspermidine could be able to disassemble an already formed and mature biofilm, a mature biofilm of 24 h was washed, added with norspermidine, incubated for 6 h and evaluated at A490 nm. Norspermidine (at 25 or 100 mM) was able to disassemble the mature biofilm in the 96 IO strain (Fig. 4A). Once again, norspermidine had not effect on the 1654 IO strain (Fig. 4B). Disassembly caused by nospermidine took place in 10 strains from a total of 31 (32.25%) at 100 mM (Table 1). The same result was observed with S. epidermidis RP62A strain. 3.5. Norspermidine inhibits S. epidermidis biofilm formation on contact lenses surface For the reason that one of the main means of causing S. epidermidis ocular infection are the contact lenses, the use of norspermidine to prevent the biofilm formation in contact lenses was assayed. Results showed that norspermidine inhibited biofilm formation on the lenses after 24 h of incubation (Fig. 5B) while biofilm was formed on non-treated lens (Fig. 5A). A graphic computational analysis shows the biofilm formation on the lenses non-treated, while the biofilm was not detectable on the lens treated with norspermidine (Fig. 5A and B). This assay was performed with three strains and the same result was obtained. 4. Discussion Polyamines are organic polycations that at physiological pH are positively charged [28]. They are involved in several cell processes, including DNA and RNA function modulation, protection against oxidative degeneration, regulation of bacterial porins and ionchannels in eukaryotic cells [29e33]. Recently, a new function of norspermidine was described acting as a biofilm formation inhibitor in B. subtilis NCBI3610, S. aureus SCO1 and E. coli MC4100 [23].
In this work was demonstrated that S. epidermidis produces and releases norspermidine into the culture medium after 40 h of incubation. Moreover, the addition of exogenous norspermidine inhibits biofilm formation. Nevertheless, the effect of norspermidine on S. epidermidis strains from IO, HS or HC was varied and different sensitivity levels to norspermidine were observed; in addition, biofilm formation in one strain from IO was not inhibited by norspermidine. This finding shows that the effect of norspermidine on biofilm formation depends on the strain and the so-called universal effect of norspemidine that Kolodkin-Gal et al. [23] showed for B. subtilis NCBI3610, S. aureus SCO1 and E. coli MC4100 strains is not accurate. This work contributes to know natural disassembly of a mature biofilm in S. epidermidis. By means of the kinetic study of biofilm formation in S. epidermidis, it was determined that the biofilm reaches the maturity after 24 h of incubation; after which, it began to be disassembled, possibly due to the production of specific compounds. This premise was supported by the inhibition of biofilm formation in young bacterial cell exposed to 40 h cell-free supernatant (Fig. 1B). However, it should not be excluded that the biofilm's disassembly at 24 h could occur due to the decrease in nutrients of the medium because the medium was not replaced. Furthermore, norspermidine was found in the 40 h cell-free supernatant and no traces of norspermidine were detected in 4 h cellfree supernatant of S. epidermidis. In another study, it was demonstrated that S. epidermidis produces D-amino acids in the stationary phase of growth and these D-amino acids also inhibit biofilm formation [27]. Thus, the results presented here suggest that after the formation of a mature biofilm, S. epidermidis produces norspermidine and D-amino acids to trigger the disassembly, and subsequent the release of the bacteria to form a new biofilm elsewhere. When norspermidine was added exogenously, biofilm formation in-vitro was inhibited; nevertheless, varying levels of sensitivity were found in the strains assayed. This suggests that S. epidermidis strains form biofilms of different biochemical composition, or with same biochemical composition but different structural arrangement. Besides, norspermidine bonds specifically to the exopolysaccharides of the biofilm of B. subtilis NCBI3610 strain [23] indicating that the target of norspermidine is exopolysaccharides. The variation in levels of sensitivity to norspermidine of the isolates tested could be explained by the following reasons: i) the different content of exopolysaccharides in the biofilms of the
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Fig. 4. Norspermidine is able to disassemble S. epidermidis biofilms. (A) S. epidermidis 96 IO strain was used to test the ability of norspermidine (Nors, 25 or 100 mM) to disassemble the biofilm. Firstly, it was allowed the biofilm formation for 24 h at 37 C in TSB (development of mature biofilm), after washing with PBS, norspermidine was added. Biofilm was measured after 6 h of incubation. (B) It shows the result for the 1654 IO strain, which was resistant to biofilm disassembly. The assays were performed six times and analyzed using a one-way ANOVA with a Tukey's test. Asterisks indicate statistical significance compared with non-treated cells.
Fig. 5. Norspermidine inhibits S. epidermidis biofilm formation on contact lenses surface. Biofilm formation was determined using either fresh TSB (A) or TSB supplemented with 25 mM norspermidine (B) on a contact lens. Images were acquired by confocal scanning laser microscopy (CSLM, Carl Zeiss Axiovert 200M) using 488 nm wavelength excitation and a 10X objective. Images were captured and processed using LSM5 Pascal software.
strains. In a previous study was demonstrated that most of the strains used in this work produce biofilm type protein- or protein/ eDNA, with a low content of exopolysaccharide [34]. However, in most of the strains, the norspermidine was required at high concentration to inhibit biofilm formation, therefore this interpretation is not viable; ii) the structural arrangement of exopolysaccharide within the biofilm; this might be the best alternative, because in these isolates the proteins or eDNA into their biofilms are the major component, and they might be covering or protecting to the exopolysaccharides toward the action of norspermidine; and finally, iii) the strains also contain eDNA within its biofilms, and norspermidine could be joined to eDNA instead of exopolysaccharides. Consequently, these results suggest that biofilms of S. epidermidis strains are complex and hence the effect of norspermidine on them is heterogenous. Previously, Kolodkin-Gal et al. [23] proposed that norspermidine is also capable to disassemble the mature biofilm of B. subtilis NCBI3610 strain. In the present work, it was found that norspermidine has a similar effect on S. epidermidis' mature biofilms, confirming the proposal by Kolodkin-Gal et al. [23]. However, the disassembly by norspermidine did not take place in all S. epidermidis strains assayed (32.25%; 10/31). To our knowledge there are not studies demonstrating the disassembly on biofilms of S. epidermidis or S. aureus by norspermidine, therefore this is the
first work that highlights this effect. The disassembly mechanism that norspermidine causes is based on its symmetrical structure (i.e. two lateral propylamines, not present in spermidine), which perfectly attaches to the negatively charged sugar residues (e.g. urionic acids), or to neutral sugars with polar groups (e.g. poly-nacetylglucosamine), causing the collapse of the exopolysaccharides and the disassembly of the biofilm [23]. In addition, we propose that norspermidine is not the sole compound acting on the biofilm, due to in some strains did not occur biofilm disassembly by norspermidine. Other compounds working in conjunction with norspermidine could disrupt biofilm's integrity. This hypothesis is based on the results obtained when the mixture of norspermidine and D-phenylalanine was assayed, and it was observed an increased inhibitory effect on biofilm formation. Furthermore, some strains may produce a more robust biofilm, which make them more resistant to the disassembly by norspermidine, and therefore such strains may have a higher level of virulence than sensitive norspermidine strains. Even though norspermidine was not able to disassemble the biofilms of all isolates, this compound may be used to cover medical equipment, in order to prevent the biofilm formation of S. epidermidis, evidenced by the inhibition assays on the surface of contact lens. Recently Hobley et al. [35] published a manuscript, which contradicts the findings reported by Kolodkin-gal et al. [23], wherein
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they exhibited that B. subtilis NCBI3610 does not produce norspermidine due to the lack of the key enzymes (carboxynorspermidine dehydrogenase, and carboxynorspermidine decarboxylase) that synthesize the compound; therefore, norspermidine could not inhibit biofilm formation [35]. We do not have an explication to Hobley's et al. [35] work, however it is worth mentioning that they did not include to Staphylococcus strains; the authors only focused on B. subtilis NCBI3610 strain, therefore their finding should not be extrapolated with the obtained for Staphylococcus strains. The findings reported in this work are similar to those obtained by Kolodkin-gal et al. [23] and support the premise that norspermidine inhibits S. epidermidis biofilm formation. However, we do not exclude that norspermidine could have a different effect to the reported here; such as in Vibrio cholerae, where norspermidine induces biofilm formation due to the effect of the NspS transmembrane protein [36]. Besides, in other work was demonstrated that norspermidine disassembles a specific type of old-aged, large microbial aggregate formed by a mixed culture [37]. Our results show clearly that the action of norspermidine on biofilms is varied and its physiological effects could depend on the bacterium and the strain assayed. In summary, in this work was demonstrated that clinical and commensal strains of S. epidermidis have different sensitivity to inhibition of biofilm formation by norspermidine, and also norspemidine is capable to disassemble a mature biofilm. This is the first study demonstrating different sensitivity levels to norspermidine by clinical and commensal strains of S. epidermidis. We propose that a mixture of norspermidine and D-amino acids may be incorporated into medical devices to prevent biofilm formation by this bacterium. Acknowledgments This work was supported by a grant from “Consejo Nacional de Ciencia y Tecnología (CONACyT) No. 153268”. FDC, ACR, SRM, MECD, JJR, and JCCD appreciate the COFAA and EDI, IPN fellowships; and support from the SNI-CONACyT. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micpath.2014.12.004. References [1] G. O'Toole, H.B. Kaplan, R. Kolter, Biofilm formation as microbial development, Annu Rev. Microbiol. 54 (2000) 49e79, http://dx.doi.org/10.1146/ annurev.micro.54.1.49. [2] G. Kogan, I. Sadovskaya, P. Chaignon, A. Chokr, S. Jabbouri, Biofilms of clinical strains of Staphylococcus that do not contain polysaccharide intercellular adhesin, FEMS Microbiol. Lett. 255 (2006) 11e16, http://dx.doi.org/10.1111/ j.1574-6968.2005.00043.x. [3] J. Wingender, M. Strathmann, A. Rode, A. Leis, H.C. Flemming, Isolation and biochemical characterization of extracellular polymeric substances from Pseudomonas aeruginosa, Methods Enzymol. 336 (2001) 302e314. [4] K.C. Rice, E.E. Mann, J.L. Endres, E.C. Weiss, J.E. Cassat, M.S. Smeltzer, K.W. Bayles, The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus, Proc. Natl. Acad. Sci. U S A 104 (2007) 8113e8118, http://dx.doi.org/10.1073/pnas.0610226104. [5] J.W. Costerton, P.S. Stewart, E.P. Greenberg, Bacterial biofilms: a common cause of persistent infections, Science 284 (1999) 1318e1322, http:// dx.doi.org/10.1126/science.284.5418.1318. [6] P.D. Fey, Modality of bacterial growth presents unique targets: how do we treat biofilm-mediated infections? Curr. Opin. Microbiol. 13 (2010) 610e615, http://dx.doi.org/10.1016/j.mib.2010.09.007. [7] J.D. Bryers, Medical biofilms, Biotechnol. Bioeng. 100 (2008) 1e18, http:// dx.doi.org/10.1002/bit.21838. [8] M. Otto, Staphylococcal biofilms, Curr. Top. Microbiol. Immunol. 322 (2008) 207e228. [9] K.L. Rogers, P.D. Fey, M.E. Rupp, Coagulase-negative staphylococcal infections, Infect. Dis. Clin. North Am. 23 (2009) 73e98, http://dx.doi.org/10.1016/
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