Effects of Extremely Low-Frequency Electromagnetic ... - Springer Link

Curr Microbiol (2010) 60:412–418 DOI 10.1007/s00284-009-9558-9

Effects of Extremely Low-Frequency Electromagnetic Fields on Helicobacter pylori Biofilm Emanuela Di Campli • Soraya Di Bartolomeo • Rossella Grande • Mara Di Giulio • Luigina Cellini

Received: 27 May 2009 / Accepted: 12 November 2009 / Published online: 24 December 2009 Ó Springer Science+Business Media, LLC 2009

Abstract The aim of this work was to investigate the effects of exposure to extremely low-frequency electromagnetic fields (ELF-EMF) both on biofilm formation and on mature biofilm of Helicobacter pylori. Bacterial cultures and 2-day-old biofilm of H. pylori ATCC 43629 were exposed to ELF-EMF (50 Hz frequency–1 mT intensity) for 2 days to assess their effect on the cell adhesion and on the mature biofilm detachment, respectively. All the exposed cultures and the respective sham exposed controls were studied for: the cell viability status, the cell morphological analysis, the biofilm mass measurement, the genotypic profile, and the luxS and amiA gene expression. The ELF-EMF acted on the bacterial population during the biofilm formation displaying significant differences in cell viability, as well as, in morphotypes measured by the prevalence of spiral forms (58.41%) in respect to the controls (33.14%), whereas, on mature biofilm, no significant differences were found when compared to the controls. The measurement of biofilm cell mass was significantly reduced in exposed cultures in both examined experimental conditions. No changes in DNA patterns were recorded, whereas a modulation in amiA gene expression was detected. An exposure to ELF-EMF of H. pylori biofilm induces phenotypic changes on adhering bacteria and decreases the cell adhesion unbalancing the bacterial population therefore reducing the H. pylori capability to protect itself. The authors Emanuela Di Campli and Soraya Di Bartolomeo contributed equally in this work. E. Di Campli S. Di Bartolomeo R. Grande M. Di Giulio L. Cellini (&) Department of Biomedical Sciences, University ‘‘G. d’Annunzio’’, Chieti-Pescara, Italy e-mail: [email protected]

123

Introduction Biofilm-forming bacteria may be considered as an ancestral selective event used by prokaryotes to adapt themselves in every environmental niche [19]. This form of mutual benefit cooperation can be considered, for the bacterial population, as the best program of survival in stressed conditions [6]. In particular, during human colonization, bacteria often prefer to live in sessile communities in order to be better organized and protected against the host defenses; in fact, it is found that an ever increasing number of infections arise from biofilm producing microorganisms [21]. Moreover, in the natural environment, biofilms can provide protection from toxic compounds, predation, and can play a role in the transmission of pathogenic bacteria [2, 20]. Helicobacter pylori is estimated to infect the gastric mucosa of half of the world’s population representing the causative agent of gastritis and peptic ulcer diseases. It has also been described as a risk factor for gastric carcinoma [28]. The natural habitat for the microorganism is the human stomach, but it may also survive in other environments, such as dental plaque, in human and animal feces [23], and in aquatic systems [1, 9]. The presence of viable cells in the environment, contributes to the levels of H. pylori in the human population also increasing its genetic variability and its environment adaptability [4, 9, 24, 26]. It has been demonstrated that H. pylori can enter the viable but not culturable (VBNC) state in which the microorganism modifies its morphology from spiral to coccoid (spherical) form with a loss of culturability [2, 7]. This cellular response to environmental stress is emphasized when bacterial cells organize themselves into microbial communities forming biofilm [2]. Therefore, the capability for this pathogen to produce a polymeric matrix forming

E. Di Campli et al.: Effects of ELF-EMF on H. pylori Biofilm

biofilm may play a very important role in its survival in the environment. The objective of this article was to investigate the role of the exposure of low-frequency electromagnetic fields (ELF-EMF), commonly produced in urban environments and in domestic appliances, on the H. pylori biofilm. The influence of electromagnetic microwaves on living organisms have been widely described resulting with different effects [5]. In particular, for prokaryotic systems, the exposure to electromagnetic fields produces stress effects causing phenotypic and transcriptional changes on free cells and affecting the surface adhesion on cells organized in biofilm [11, 13, 18]. In this report, the effect of ELF-EMF both on the formation and on the detachment of the H. pylori mature biofilm was evaluated. To do this, biofilms were exposed to ELF-EMF during formation and after maturity, then compared to their respective sham exposed controls for: (i) the cell viability status; (ii) the cell morphological aspects; (iii) the biomass measurement; (iv) the analysis of DNA fingerprintings and the expression of luxS and amiA genes coding for a significant indicator of biofilm production and a coccoid transforming protein, respectively.

Materials and Methods Exposure System and Field Characteristic The magnetic fields were generated by a cylindrical solenoid, measuring 170 mm in diameter internally, with a length of 450 mm with 180 turns of copper wire (Oersted Technology Corp., Oregon, USA). This device was designed and built to deliver variable, homogeneous, sinewave alternating magnetic fields regulated and defined along the center line with 50 Hz frequency and intensities ranging between 0.1 and 1.0 mT ±2%. The polarization of the field generated was vertical and the solenoid was able to ensure the maximum homogeneity (within 1%) in a cylindrical volume with radius of 60 mm and length of 190 mm, centered in the middle of the solenoid. The solenoid was powered by an Elgar series CW power supply (Elgar Electronics, San Diego, California, USA) capable of forming a sine-wave output with very little distortion at a closely regulated frequency and voltage (or current), regardless of distortions, frequency and voltage variations of the line service. In particular, the power supply was operated in its ‘‘closed-loop current mode’’: the output current and the generated magnetic field were consequently regulated against thermal and physical variations/drift of the coil. The final measured value of the solenoid coil constant was 4.630 Gauss/Amp. A magnetic flux density of 1 mT was generated at 50 Hz. This parameter was selected

413

since it is a common exposure in urban environments and in domestic appliances. The analyzed samples, contained in systems transparent to the ELF-EMF and put in microaerophilic atmosphere (Campy Gen Compact—Oxoid; Milan, Italy), were located on a non-magnetic support in the maximum homogeneity part of the magnetic field inside the coil system. The solenoid was vertically placed into an incubator (Heraeus 5042E, Hamburg, Germany) at 37°C. The same experimental design, planned for the active coil, was performed by switching off the coil in order to compare differences between the biofilm formation and the mature biofilm in exposed and in the sham exposed samples corresponding to the controls. Bacterial Strains and Experimental Design The international reference strain H. pylori ATCC 43629 was used for the experiments. The strain, stored at -80°C, was rapidly plated on Chocolate agar (CA) plus 1% of IsoVitaleX (Becton–Dickinson, Buccinasco, Italy) and incubated at 37°C for 5–7 days in microaerophilic atmosphere consisting of 85% N, 5% O2, and 10% CO2 (Rivoira, Milan, Italy). Then, bacteria were harvested in Brucella broth (BB, Biolife Italiana, Milan, Italy) supplemented with 2% (w/v) of fetal calf serum (Biolife Italiana) and 0.3% (w/ v) of glucose (Sigma–Aldrich, Milan, Italy). Broth cultures, shaken at 120 rpm in a water bath incubator (Julabo SW20C incubator, Johns Scientific, Inc., Toronto, Canada) were incubated overnight at 37°C in microaerophilic atmosphere (85% N, 5% O2, 10% CO2). After incubation, each broth culture was adjusted to an optical density at 600 nm (OD600) of 0.1 and used for the experiments. In particular: 2 ml were inoculated in 3.5 cm polystyrene Petri discs (Steroglass, Perugia, Italy) for the viability test and the morphology detection; 200 ll of the cell suspension were inoculated in flat-bottomed 96-well treated polystyrene microtiter plates (Nunc, EuroClone SpA, Life-SciencesDivision, Milan, Italy) for the determination of the biofilm formation; and 12 ml were inoculated in cell culture polystyrene flasks of 25 cm2 with 0.2 lm vent cap (Corning, New York, USA) for the molecular analysis. The effects of exposure to ELF-EMF of H. pylori biofilm was studied in two different experimental conditions: (a) during the biofilm formation and (b) on the mature biofilm. (a)

ELF-EMF on the biofilm formation: all the prepared culture samples were exposed to 50 Hz frequency and 1 mT magnetic field for 2 days at 37°C in microaerophilic atmosphere (Campy Gen compact—Oxoid; Italy). (b) ELF-EMF on the mature biofilm: all the prepared culture samples were previously grown as biofilms for 2 days in the switched off coil at 37°C in

123

414


microaerophilic atmosphere and then, as above, exposed to 50 Hz frequency and 1 mT magnetic field for 2 days at 37°C in microaerophilic atmosphere, for a total time of 4 days of incubation. For control, sham exposed sessile cultures of 2- and 4-day-old were incubated in the switched off coil at 37°C in microaerophilic atmosphere, and were checked and compared to the corresponding exposed cultures. The experimental design was carried out in active and sham coils for five independent experiments and each experiment was performed in triplicate. Viability Test and Microscopic Observations The H. pylori biofilm, produced by experimental condition (a) and (b), was mounted on 3.5 cm Petri discs and examined for their viability with Live/Dead Kit (Molecular Probes Inc., Invitrogen, Milan, Italy) as per the manufacturer’s instructions and visualized under a fluorescent Leica 4000 DM microscope (Wetzlar, Germany). As for the bacterial shape, the number of spiral (S) and coccoid (C) forms was determined by counting ten randomly chosen fields of view. Three microbiologists repeated the counts independently. The detected U shaped bacteria were considered as coccoid cells. Biofilm Assay The effect of ELF on H. pylori biofilm, produced by experimental condition (a) and (b), was evaluated by determination of adhesion to polystyrene flat-bottomed microtiter plates and was quantified by safranin staining and reading the absorbance at 492 nm. After cultures incubation, for quantitative measurements, a modified method of Cramton et al. [16] was used. Briefly, the planktonic bacteria were removed by aspiration and each well was washed twice with phosphate buffer solution (PBS), dried and stained with a 0.1% safranin solution for 1 min and then washed with water. The optical density (OD) of stained biofilms was measured at 492 nm using an enzyme-linked immunoadsorbent assay (ELISA) reader (SAFAS, Munich, Germany).

pylori deoxynucleotide primer 1290 (60% G–C) was used in a low stringency PCR amplification (2700 thermocycler; P.E. Applied Biosystems, Foster City CA, USA) following the methodology reported by Cellini et al. [8]. Detection of luxS and amiA Genes Expression For the analysis of the expression of luxS and amiA genes, H. pylori biofilm, produced by experimental condition (a) and (b), was removed using a cell scraper (Corning). Bacterial RNA was extracted from sessile cells using RnasyMini Kit (Qiagen). Before performing the Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), a DNase digestion was performed. The glmM gene, expressed constitutively in H. pylori, was inserted as internal control and included in Multiplex RT-PCR (MRT-PCR) with the luxS gene. The RT-PCR and MRT-PCR were determined by using luxS, amiA, and glmM primers described elsewhere [3, 17, 22]. Both MRT-PCR and RTPCR were performed using One Step RT-PCR Kit (Qiagen) in a final reaction volume of 25 ll containing: 70 ng of total RNA, 19 Qiagen buffer, 500 lM of each dNTP, 0.6 lM of each primer, and 5 U of One Step RT enzyme. RT-PCR Master Mix was performed in a 2700 thermocycler (Applied Biosystems) for 30 min at 50°C and 15 min at 95°C for retro-transcription and initial PCR activation, respectively. Amplification of luxS and glmM cDNA consisted of 95°C for 15 min and then 30 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 1 min, with a final 10 min extension at 72°C. The same procedure was used for amiA amplification, except for the annealing temperature which was 64°C. Samples (6 ll) of PCR products were analyzed by electrophoresis in a 2% (w/v) agarose gel at 100 V for 45 min. Gels were stained with ethidium bromide (Boehringer Mannheim, GmbH, Germany) and images were acquired by Gel Doc XR (Bio-Rad, Laboratories, srl, Milan, Italy). Statistical Analysis The evaluation of the differences statistically significant (P \ 0.05) recorded in the experiments performed with and without ELF-EMF exposure was performed by means of ANOVA followed by Dunnett’s t test.

Random Amplified Polymorphic DNA (RAPD) Fingerprinting Detection Results Helicobacter pylori chromosomal DNA was extracted from the biofilm, produced by experimental condition (a) and (b), using QIAamp Tissue DNA isolation minikit (QIAGEN S.p.A., Milano, Italy). Sessile bacteria were removed by using a cell scraper (Corning, Corning Incorporated Life Sciences, NY, USA). For RAPD-PCR, the specific H.

123

To verify the effect of 50 Hz–1 mT EMF intensity on the biofilm formation and on the mature biofilm of H. pylori, bacterial cultures as well as on 2-day-old H. pylori ATCC 43629 biofilm, a 2 days exposure and a comparison to the respective sham exposed controls was completed.


The bacterial morphology recorded on cells during the biofilm formation, a significant difference in bacterial morphology was found between sham and exposed sessile bacteria (Fig. 1a). During the biofilm formation, the H. pylori cells attached to polystyrene surface under 50 Hz and 1 mT exposure, retained the spiral morphology (58.4% ± 7.2 SEM) compared to the sham exposed cells (33.1% ± 14.2 SEM). The cultures showed significant differences in cell viability (Fig. 2). Helicobacter pylori cells averaged 58.9% ± 6.3 viable cells among attached exposed cultures, while sham exposed cultures averaged 85.3% ± 11.8 viability (Fig. 1a).

Fig. 1 Effect of ELF-EMF (50 Hz of frequency and 1 mT of intensity) on morphology (S, spiral, C, coccoid) and on viability of Helicobacter pylori ATCC 43629 biofilm. The ELF-EMF effect was evaluated on: (a) biofilm formation and (b) on mature biofilm. The results presented here are the mean values ± SEM (Standard Error of the Media) of five independent experiments performed in triplicate

415

Figure 2 shows representative images of H. pylori cells during the biofilm formation exposed (Fig. 2a) and sham exposed (Fig. 2b) to ELF-EMF for 2 days. Mixed live and dead cells were observed in exposed cultures in which the presence of the U shaped morphology (Fig. 2a, insert) was a common aspect detected in each counted field. When the aspects discussed above were examined on mature biofilm, no significant difference was recorded with regard to both the bacterial morphology and the cell viability, in respect to the sham exposed controls of 4 days (Fig. 1b). In this experimental condition, H. pylori sessile cultures were characterized by cells with a prevalent coccoid morphotype (70.1% ± 5.5 and 77.4% ± 7.4 SEM in exposed and sham exposed cultures, respectively). Among the detected coccoid cells, the percentage of viability was 34.84% and 36.6% for the exposed and sham exposed cultures. The measurement of biofilm cell mass of H. pylori, quantified by safranin staining, displayed a significant effect of ELF-EMF in the inhibition of biofilm formation and a reduction in adhesion to a polystyrene surface in respect to the sham exposed samples (Table 1). When the exposed sessile cultures and the corresponding sham controls were compared for their DNA fingerprintings to evaluate the presence of micro and macroevolutions, no significant differences among DNA patterns were recorded for each examined condition (Fig. 3a). With regard to the expression of the luxS gene, no notable differences were observed between exposed and sham exposed samples during the biofilm formation and on mature biofilm (Fig. 3b). On the contrary, the product of

Fig. 2 Representative images of Live/Dead viability stain of Helicobacter pylori ATCC 43629 cells during the biofilm formation. (a) Sessile culture exposed to ELF-EMF compared to (b) sham exposed sessile culture. The insert displays the widespread presence of Ushaped morphology among the exposed bacterial population. Original magnification 91000. Scale bars = 5 lm

123

416


Table 1 Measurement of biofilm cell mass of Helicobacter pylori ATCC 43629 exposed at a frequency of 50 Hz and 1 mT of electromagnetic fields (ELF-EMF) Experimental condition

Biofilm cell mass measurement (absorbance at OD492) Exposed

Sham exposed

Biofilm formation

0.0067 (±0.0036)

0.0183 (±0.0060)

Mature biofilm

0.1472 (±0.0394)

0.2759 (±0.0678)

The values (mean ± standard deviation) presented here are representative of those obtained in five experiments; each experiment was performed in triplicate

Fig. 3 Representative DNA fingerprintings (a) and expressions of luxS/glmM genes (b) and the amiA gene (c) of Helicobacter pylori ATCC 43629 biofilm exposed to ELF-EMF. The ELF-EMF effect was evaluated on H. pylori cells during the biofilm formation (lanes 1: exposed samples, lanes 2: sham exposed samples) and on mature biofilm (lanes 3: exposed samples, lanes 4: sham exposed samples). DNA size standards (0.1 kbp Marker) are in the lane marked M. The sizes of the amplification products were approximately 465, 252, and 425 base-pairs (bp) for luxS, glmM, and amiA, respectively. The results shown here, are representative of results obtained in five independent experiments performed in triplicate

the amiA gene was expressed at higher levels in the biofilm formation (particularly in the sham exposed samples) with respect to the mature biofilm (Fig. 3c). At each time point, glmM, included as an internal control with luxS (Fig. 3b), was always expressed.

Discussion The biofilm formation by pathogen bacteria in the environment can represent a protected ecological niche, functional for bacterial transmission [1, 21]. Helicobacter pylori is able to form a biofilm to protect itself [10, 14] and the biofilm is able to persist in water biofilms [20],

123

therefore overcoming the stressful environmental condition and favoring bacterial transmission. In this study, the effect of 50 Hz and 1 mT both on the cell adhesion and on the H. pylori mature biofilm was evaluated. It was found that ELF-EMF interfere with sessile H. pylori morphology during the biofilm formation. In particular, during the biofilm formation the exposed bacterial population retained the spiral form in comparison with the coccoid form found in sham exposed bacteria. In fact, the exposed biofilm displayed a reduced number of coccoid cells, not completely rounded which were less viable in respect to the corresponding controls. Interestingly, the higher standard error of the mean displayed only in the sham exposed controls underlines the extraordinary fickleness of this microorganism less capable to display its variability in stressed conditions such as ELF-EMF exposure. The difference in the morphological aspect was confirmed in cultures after 2 days of exposure by a weaker expression of the amiA gene, involved in the transition from spiral to coccoid H. pylori morphology [12]. As a consequence, the major presence of spiral cells in the exposed samples, may facilitate the ELF-EMF damage of membranes; therefore, favoring the penetration of fluorescent propidium iodide resulting in red dead spiral cells. Previous studies demonstrated that the morphological transition for H. pylori is related to a more protected condition in which the coccoid cells are characterized both by producing a peptidoglycan similar to those of sporulating Bacillus sphaericus [15] and being able to escape detection by the immune system [12]. On the basis of these observations, our findings suggest that an ELF-EMF exposure may act by unbalancing the sessile H. pylori population; the adhered cells became unable to activate their adaptative machinery resulting in a decrease in coccoid morphotypes and in cell viability. Regarding the biofilm formation, a reduction in cell biomass was found in cultures during the biofilm formation, in respect to the controls. Similarly, Chua and Yeo [13] demonstrated that the surface bio-magnetism, through perpendicularly polarized magnetic media was able to influence the bacterial adhesion as well as the protein synthesis. These authors pointed to a new understanding in biophysics of cell adhesion also detecting modified cells with a swollen aspect. No differences in viability and cell morphology between exposed and sham exposed biofilms were found, with regard to the effect of ELF-EMF on mature biofilms, but, the measurement of cell biomass attached to polystyrene revealed a significant reduction on adhesion. In this experimental condition, the electromagnetic field did not interfere on the bacterial morphotype but resulted effective in the controlling of cell adhesion as observed during the biofilm formation.


The exposed cultures and the corresponding controls were also studied for their DNA profiles to evaluate possible micro or macro evolutions induced by static magnetic field. No differences were detected in DNA fingerprintings coming from exposed and sham exposed samples. This data were consistent with our previous investigation on Escherichia coli ATCC 700926 [11] and with the results obtained by Potenza et al. [25] that found a genoma stability among exposed and unexposed Escherichia coli XL-1. With regard to the luxS gene, involved in QuorumSensing mechanisms and in biofilm formation, a similar expression was found in each experimental condition examined both in exposed and sham exposed cultures underling a cross-talking activity among the sessile H. pylori population. On the other hand, Trushin [29] ascribes to electromagnetic fields a role in the microbial biocommunication and Shcheglov et al. [27] found a dependence on bacterial cell density facilitating a cell–cell communication during response to ELF. In conclusion, an exposure of 50 Hz and 1 mT modifies the H. pylori organization both on bacterial morphology and on cell adhesion; bacteria become less capable of progressing in their viable persistent morphology also reducing their clustered state. Additional studies should be performed to better understand the different aspects of environmental microbial life, including bacterial adhesion and bacterial response to stressing factors such as ELFEMF. Acknowledgements The Authors thank Rosa Conese for her technical assistance. This study was supported by a grant awarded by the ‘‘Ministero Universita` e Ricerca’’, PRIN 2008, Rome, Italy.

References 1. Adams BL, Bates TC, Oliver JD (2003) Survival of Helicobacter pylori in a natural freshwater environment. Appl Environ Microbiol 69:7462–7466 2. Andersen LP, Rasmussen L (2009) Helicobacter pylori-coccoid forms and biofilm formation. FEMS Immunol Med Microbiol 4:1–4 3. Aras RA, Kang J, Tschumi AI, Harasaki Y, Blaser MJ (2003) Extensive repetitive DNA facilitates prokaryotic genome plasticity. Proc Natl Acad Sci USA 100:13579–13584 4. Azevedo NF, Guimara˜es N, Figueiredo C, Keevil CW, Vieira MJ (2007) A new model for the transmission of Helicobacter pylori: role of environmental reservoirs as gene pools to increase strain diversity. Crit Rev Microbiol 33:157–169 5. Berg H (1999) Problems of weak electromagnetic field effects in cell biology. Bioelectrochem Bioenerg 48:355–360 6. Branda SS, Kolter R (2004) Multicellularity and biofilms. In: Ghannoum M, O’Toole GA (eds) Microbial biofilms. ASM Press, Washington DC, pp 20–27 7. Cellini L, Robuffo I, Di Campli E, Di Bartolomeo S, Taraborelli T, Dainelli B (1998) Recovery of Helicobacter pylori

417

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

ATCC43504 from a viable but not culturable state: regrowth or resuscitation? APMIS 106:571–579 Cellini L, Di Campli E, Di Candia M, Marzio L (2003) Molecular fingerprinting of Helicobacter pylori strains from duodenal ulcer patients. Lett Appl Microbiol 36:222–226 Cellini L, Di Campli E, Grande R, Di Bartolomeo S, Prenna M, Pasquantonio MS, Pane L (2005) Detection of Helicobacter pylori associated with zooplankton. Aquatic Microb Ecol 40:115–120 Cellini L, Grande R, Traini T, Di Campli E, Di Bartolomeo S, Di Iorio D, Caputi S (2005) Biofilm formation and modulation of luxS and rpoD expression by Helicobacter pylori. Biofilms 2:1–9 Cellini L, Grande R, Di Campli E, Di Bartolomeo S, Di Giulio M, Robuffo I, Trubiani O, Mariggio` MA (2008) Bacterial response to the exposure of 50 Hz electromagnetic fields. Bioelectromagnetics 29:302–311 Chaput C, Ecobichon C, Cayet N, Girardin SE, Werts C, Guadagnini S, Pre´vost MC, Mengin-Lecreulx D, Labigne A, Boneca IG (2006) Role of AmiA in the morphological transition of Helicobacter pylori and in immune escape. PLoS Pathog 2:e97 Chua LY, Yeo SH (2005) Surface bio-magnetism on bacterial cells adhesion and surface proteins secretion. Coll Surf B Biointer 40:45–49 Cole SP, Harwood J, Lee R, She R, Guiney DG (2004) Characterization of monospecies biofilm formation by Helicobacter pylori. J Bacteriol 186:3124–3132 Costa K, Bacher G, Allmaier G, Dominguez-Bello MG, Engstrand L, Falk P et al (1999) The morphological transition of Helicobacter pylori cells from spiral to coccoid is preceded by a substantial modification of the cell wall. J Bacteriol 181:3710– 3715 Cramton SE, Gerke C, Schnell NF, Nichols WW, Gotz F (1999) The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infect Immun 67:5427–5433 De Reuse H, Labigne A, Mengin-Lecreulx D (1997) The Helicobacter pylori ureC gene codes for a phosphoglucosamine mutase. J Bacteriol 179:3488–3493 Del Re B, Bersani F, Agostini C, Mesirca P, Giorgi G (2004) Various effects on transposition activity and survival of Escherichia coli cells due to different ELF-MF signals. Radiat Environ Biophys 43:265–270 Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15:167–193 Giao˜ MS, Azevedo NF, Wilks SA, Vieira MJ, Keevil CW (2008) Persistence of Helicobacter pylori in heterotrophic drinkingwater biofilms. Appl Environ Microbiol 74:5898–5904 Jain A, Gupta Y, Agrawal R, Khare P, Jain SK (2007) Biofilms— a microbial life perspective: a critical review. Crit Rev Ther Drug Carrier Syst 24:393–443 Joyce EA, Bassler BL, Wright A (2000) Evidence for a signaling system in Helicobacter pylori: detection of a luxS encoded autoinducer. J Bacteriol 13:3638–3643 Kabir S (2003) Review article: clinic-based testing for Helicobacter pylori infection by enzyme immunoassay of faeces, urine and saliva. Aliment Pharmacol Ther 17:1345–1354 Lu Y, Redlinger TE, Avitia R, Galindo A, Goodman K (2002) Isolation and genotyping of Helicobacter pylori from untreated municipal wastewater. Appl Environ Microbiol 68:1436–1439 Potenza L, Cucchiarini L, Piatti E, Angelini U, Dacha M (2004) Effects of high static magnetic field exposure on different DNAs. Bioelectromagnetics 25:352–355 Schwarz S, Morelli G, Kusecek B, Manica A, Balloux F, Owen RJ, Graham DY, van der Merwe S, Achtman M, Suerbaum S

123

418 (2008) Horizontal versus familial transmission of Helicobacter pylori. PLoS Pathog 4:e1000180 27. Shcheglov VS, Alipov ED, Belyaev IY (2002) Cell-to-cell communication in response of E. coli cells at different phases of growth to low-intensity microwaves. Biochim Biophys Acta 1572:101–106

123

E. Di Campli et al.: Effects of ELF-EMF on H. pylori Biofilm 28. Suerbaum S, Michetti P (2002) Helicobacter pylori infection. N Engl J Med 347:1175–1186 29. Trushin MV (2003) Studies on distant regulation of bacterial growth and light emission. Microbiology 149:363–368