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Experimental Physiology 90.6 pp 855-863
DOI: 10.1113/expphysiol.2005.030858
© The Physiological Society 2005
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Extremely low electrical current generated by porcine small intestine smooth muscle alters bacterial autolysin production

Danuta Kruszewska1, Pawel Podgurniak2, Åsa Ljungh1, Aleksandra Sebastian1, Lennart Larsson1, Jolanta Zajdel-Dabrowska3 and Stefan G Pierzynowski45

1 Department of Laboratory Medicine, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden 2 Department of Physiological Sciences, Warsaw Agricultural University, Nowoursynowska 159, 02-787 Warsaw, Poland 3 Department of Pharmaceutical Microbiology, Medical University, Oczki 3, 02-007 Warsaw, Poland 4 Department of Cell and Organism Biology, Lund University, Helgonavägen 3b, SE-223 62 Lund, Sweden 5 Sea Fisheries Institute, Kollataja 1, 81-332 Gdynia, Poland


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The effect of extremely low electrical currents, identical to those generated by the gut smooth muscle, on bacterial autolysin production in vitro was tested in the present study. When stimulated with these electrical currents, the bacteria Pediococcus pentosaceus 16:1 produced groups of peptidoglycan hydrolases that differed from those produced by the unstimulated (control) bacteria. The autolysins synthesized by the P. pentosaceus 16:1 under extremely low electrical currents were effective against peptidoglycans from the cell walls of various lactic acid bacteria strains, whereas the autolysins from the control bacteria acted exclusively against P. pentosaceus 16:1 cell walls. Thus, it can be predicted that in vivo the electrical currents generated by the intestinal smooth muscles, which can be recorded as the myoelectrical migrating complexes, could regulate lactic acid bacteria strain growth in the gut.

(Received 8 May 2005; accepted after revision 12 August 2005; first published online 23 August 2005)
Corresponding author : S. G. Pierzynowski: Department of Cell and Organism Biology, Lund University, Helgonavägen 3b, SE-223 62 Lund, Sweden. Email: stefan.pierzynowski{at}telia.com


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Lactic acid bacteria (LAB) are now widely used as probiotics. Autolysins, which are endogenous enzymes in these bacteria, hydrolyse specific bonds in peptidoglycans within the bacterial wall, and in this way can regulate their growth and that of other bacteria. A wide variety of these enzymes has been identified, including N-acetylmuramidases (lysozymes), N-acetylglucosaminidases, N-acetylmuramyl-L-alanine amidases, endopeptidases and transglycosylases (Schleifer & Kandler, 1972). The beneficial health effects of the LAB used as probiotics should be exhibited after the colonization of the small intestine. However, during the colonization process the LAB, particularly those adhering to the enterocytes, are exposed to the extremely low electrical currents (ELEC) generated by the intestinal smooth muscle. It has been shown that ELEC similar to those generated by the smooth muscle affect bacterial growth in vitro (Grzesiuk et al. 2001; Wojcik-Sikora et al. 2001). The smooth muscle myoelectrical activity precedes its mechanical action, which in turn will influence the flow (mixing and propelling) of the digesta. The myoelectric activity consists of the electrical control activity (ECA or slow waves) and the electrical response activity (ERA or spike activity). The ECA is an omnipresent oscillation of membrane potential, generated by a network of the interstitial cells of Cajal and spread electronically to the smooth muscle cells. This controls the excitability of the smooth muscles and largely controls the frequency of contraction, whereas the ERA is under the influence of regulatory mechanisms and determines the contractile force (Tomita, 1981; Sarna, 1989; Sanders, 1996; Laerke et al. 2000).

The main objective of this study was to test whether ELEC, having characteristics which are the same as those of the ECA and ERA, and manifesting as the myoelectrical migrating complexes (MMC) of the pig small intestine, under in vitro conditions could affect the production of peptidoglycan hydrolases by Pediococcus pentosaceus 16:1, a member of the LAB able to colonize the intestine.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Preliminary preparation of bacteria

Preparation of bacterial cultures and cell walls.  Four species of LAB, namely P. pentosaceus 16:1, Lactobacillus plantarum 2592, Lactobacillus paracasei ssp. paracasei F19 and Leuconostoc mesenteroides 77:1, were used in this study. P. pentosaceus 16:1 and L. mesenteroides 77:1 were derived from fermented organically cultured rye, whereas the lactobacillae were isolated from human large intestinal mucosa (from the collection of Lund University, Department of Medical Microbiology, Dermatology and Infection; Kruszewska et al. 2002). The LAB isolates were cultured in de Man Rogosa-Sharpe (MRS) broth (Oxoid) for 24 h at 37°C under microaerobic conditions.

The cell walls of P. pentosaceus 16:1, L. plantarum 2592, L. paracasei ssp. paracasei F19 and L. mesenteroides 77:1 were separated after growth in the MRS broth, supplemented with 0.03 M glucose at 35°C to an optical density (OD) at 625 nm of 1.0 (exponential phase). The cells were harvested by centrifugation at 10 000g for 15 min at 4°C, and the pellet obtained was washed twice with ice-cold 0.01 M potassium phosphate buffer (PBS; pH 6.5) and resuspended in 2 ml of ice-cold distilled water (pH 5.5–6.0). In order to prepare cell wall extracts, 2 ml of 8% of SDS was added to these cell suspensions and the mixture was boiled for 30 min at 100°C. After boiling, the suspension was adjusted to 16 ml with 4% SDS. The extraction was continued overnight at room temperature (RT) with gentle stirring. After centrifugation at 10 000g for 15 min at 4°C the supernatant was harvested. The pellets were resuspended as described above and SDS extraction was repeated. The pellets were washed (a minimum of 10 times) with 0.01 M Tris-HCl (pH 6.5) and then dialysed against frequent changes of distilled water at 4°C and suspended in 0.6 ml of distilled water (Massidda et al. 1996).

Crude peptidoglycan preparation.  Crude cell walls were treated with 0.1 N HCl at 60°C for 16 h. The suspension was neutralized with 2.5 N NaOH. The peptidoglycan was collected after centrifugation at 20 000g for 20 min, and the pellet was washed five times with 0.01 M Tris-HCl (pH 6.5). The detailed procedure for the determination of muramic acid is described elsewhere (Sebastian & Larsson, 2003). In brief, samples were heated in 2 M methanolic HCl at 85°C overnight, after which the internal standard (C13-labelled MuAc in a methanolysate of C13-labelled algal cells) was added. The mixture was extracted with heptane, and the lower phase was further purified using propylsulphonic acid columns, evaporated to dryness under nitrogen and further dried under vacuum in a desiccator (2 h). The samples were then acetylated by heating in a mixture of pyridine and acetic anhydride at 60°C for 1 h. The reaction mixture, dissolved in dichloromethane, was subsequently washed, first with a 0.05 M HCl solution and thereafter with water. The samples were then purified using silica gel columns, evaporated to dryness, dissolved in chloroform and analysed using AN ion trap gas chromatograph/mass spectrometer (GC-MSMS). Sample preparation resulted in methylation of the carboxyl and anomeric hydroxyl groups and acetylation of the three remaining hydroxyl groups. Fragmentation of mass to charge (m/z) 187 led to a high intensity of the product ion m/z 145, which was therefore monitored in the MSMS mode.

Electrical stimulation of P. pentosaceus 16:1

ELEC treatment and characteristics.  The experiments were carried out in glass bacteriological tubes of 15 mm internal diameter containing 10 ml of bacterial suspension (106 colony forming units (cfu) ml–1). The facing parallel platinum plate electrodes, 5 mm wide and 0.5 mm thick, were inserted from the top to the bottom of the tubes 10 mm apart and connected to the output of a GGP-3 MMC Simulator (Flow Instruments, Warsaw, Poland) which was used to generate a typical ELEC (both ECA and ERA) of the porcine intestine. The strength of the generated electric field was Epeak = ±83 µV mm–1, average conductivity of the medium {sigma} = 15 mS cm–1 (Gu & Justiz, 2002) and average sample resistance R = 10 {Omega}. The calculated maximal current density during a mean simulated spike potential was jpeak = ±8.3 µA cm–2. The characteristic output voltages of the device, which are the first derivatives of the spreading transmembrane potential changes of the porcine ileum (Jimenez et al. 1999), are presented in Figs 1 & 2. The upper trace in Fig. 1 shows the typical output voltage of the simulator during the slow waves of the phase I of the myoelectrical migrating complexes (MMC; ECA only, no spikes), and the lower trace shows the typical output voltage during the slow waves with the superimposed action potentials of the smooth muscles (ECA + ERA) of phase III of the MMC (Lammers & Slack, 2001). The long-term periodical changes of stimulated ERA activity forming the typical pattern of the porcine MMC are shown in Fig. 2 (Bueno et al. 1982; Fleckenstein et al. 1982).



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  		Figure 1.  Oscillograms of the GGP-3 MMC Simulator output waveforms
Upper trace shows slow waves with no action potentials during phase I of the MMC. Lower trace shows slow waves with action potential activity during phase III of the MMC. Calibration: 2 s per division and 20 mV per division.

 


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  		Figure 2.  Periodical changes of the ERA activity in the simulated MMCs
The histogram shows GGP-3 MMC Stimulator output. cpm, counts per minute.

 
Cellular autolysis.  The autolytic phenotype of the P. pentosaceus 16:1 strain was evaluated under starvation conditions for 72 h, in 0.01 M PBS (pH 6.8) at 30°C with or without exposure to the ELEC. Culture conditions, such as time, temperature, osmolarity and pH, were experimentally determined.

The culture of P. pentosaceus 16:1 in an exponential phase was rapidly chilled (ice-bath). The cells were harvested by centrifugation at 10 000g for 15 min at 4°C and washed three times with distilled water at 4°C. The bacterial pellet was resuspended in 10 ml of PBS, to an OD at 625 nm of approximately 1.0 (106 cfu ml–1; data not shown), and the suspensions were incubated in a 30°C water bath for 72 h. Six samples were always examined simultaneously. The ELEC was generated exclusively in three tested samples. In the remaining three samples with the same experimental set-up, the current was not generated and these samples served as a control. The studies were repeated six times. In all tested samples, the degree of autolysis was expressed as a decrease in the OD at 625 nm, calculated as the extent of the turbidity in the tested samples. OD values were measured every hour for the first 12 h, and every 3 h thereafter for the remaining 60 h (Lortal et al. 1997). Following the above tests, the cultures were centrifuged at 10 000g for 15 min at 4°C, and the cell-free supernatants were concentrated for further studies, as described below.

Identification of peptidoglycan hydrolases using renaturing two-dimensional gel electrophoresis (2-DE)

In order to identify the number of peptidoglycan hydrolases released by P. pentosaceus 16:1 exposed to starvation stress for 24 h and treated or untreated with the ELEC, the 2-DE was run with one repetition for each supernatant obtained from each of six ELEC-treated and six untreated bacterial suspensions. The P. pentosaceus 16:1 peptidoglycan was used as a target for the enzymatic activity (Kornilovs'ka et al. 2002; Kaino et al. 1998).

Isoelectric focusing (IEF) was performed using IPGphorTM horizontal electrophoresis (Pharmacia Biotech, Stockholm, Sweden). A series of three to ten immobilized pH gradient strips (7 cm long; Amersham Bioscience, USA) were rehydrated in 0.25 ml sample buffer range 3–10 (Amersham Bioscience, USA) containing 8 M urea, 2% 3-((3-cholamidopropyl)-dimethylammonio)-1-propane-sulphonate (CHAPS), 1% dithiothreitol (DTT) and the proteins to be tested. IEF was performed according to the manufacturer's instructions.

Before renaturing, the SDS-PAGE first dimensional immobilized pH gradient (IPG) strips were equilibrated twice in solutions containing mainly DTT and iodoacetamide, respectively. The strips were then applied to 12% gels supplemented with P. pentosaceus 16:1 peptidoglycan (0.1% w/v), and analysed under reducing conditions, where separation was conducted at 150 V for 60 min. Following electrophoresis, the substrate-containing gels were incubated in distilled water at RT for 30 min and then placed into the renaturation buffer (0.05 M Tris-HCl buffer, pH 6.8, containing 0.1 M KCl and 0.1% Triton X 100). After a second 30 min incubation at RT, the gels were transferred to fresh renaturating buffer and the process was run for 16 h at 37°C. The lytic activity of the hydrolases was visualized as clear spots against a light blue background after gel staining with 0.1% Methylene Blue in 0.01% KOH for 1 h (Lorca et al. 2001).

SDS-PAGE gels were scanned with a GS-710 Imaging Densitometer (Bio-Rad; USA), and the molecular weights and isoelectric potential (pI) of the proteins were estimated by image analysis using the Melanie V3 software package (Bio-Rad, USA).

Peptidoglycan hydrolases obtained from the autolysis of P. pentosaceus 16:1 and bacterial cell walls

The degradative activity of the P. pentosaceus 16:1 peptidoglycan hydrolases obtained after treatment was evaluated against the cell walls of the lactic acid bacteria L. plantarum 2592, L. paracasei ssp. paracasei F19 and L. mesenteroides 77:1 obtained as described above. Briefly, native cells and cell walls derived from the lactic acid bacteria were autoclaved at 120°C for 15 min. Then, the cells and cell walls were disrupted by ten ultrasonication cycles (20 s each) at 4°C with 20 s intervals. For native cell wall separation, a two step centrifugation procedure was applied.

The pellets containing native cells of P. pentosaceus 16:1 (0.4% w/v), L. plantarum 2592 (0.4% w/v), L. paracasei F19 (0.4% w/v), L. mesenteroides 77:1 (0.4% w/v) and Micrococcus lysodeikticus ATCC 4698 (0.2% w/v; Sigma, USA; positive control), respectively, were washed with cold water and incorporated into 1% agarose (Litex, Denmark) in PBS (pH 6.8) on Petri dishes (Strating & Clarke, 2001). The supernatants from the ELEC-treated and untreated suspensions of P. pentasaceus 16:1 (20 µl) were poured into agarose gels enriched with the native cell walls obtained from the lactic acid bacteria. Lysosyme was used as the positive control. Hydrolytic activity was measured as the diameter (in mm) of transparency in the agarose gels caused by degradation of the lactic acid bacterial cell walls by P. pentosaceus 16:1 peptidoglycan hydrolases.

Statistics

Since the criteria of normality of distribution and the equality of the variability of data were fulfilled (as indicated by a {chi}2 test), parametric tests were used. The results of the hydrolase activity in the supernatant obtained from P. pentosaceus 16:1 suspensions treated and untreated with ELEC were compared statistically using Student's unpaired t test or a two-way analysis of variance (ANOVA) and Tukey's post hoc test (SigmaStat for Windows v2.0, SPSS Science, Chicago, IL, USA).


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Experimental conditions

A phosphate concentration of 0.01 M at pH 7.2, room temperature (25°C) was found to promote greater autolysis of P. penasaceus 16:1 compared to a phosphate concentration of 0.1 M at pH 6.8 (P < 0.01). Temperatures within the range 30–40°C also increased the autolytic responses (P < 0.01; Fig. 3). However, a tendency (P < 0.057) to a greater degree of autolysis was observed in the ELEC-treated samples when they were incubated at 30°C. The above observations were the main factors determining the experimental conditions for the entire experiment, which were set as phosphate concentration 0.01 M and temperature 30°C.



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  		Figure 3.  The degree of autolysis of the P. pentosaceus 16:1 cells after 10 h incubation with ({blacksquare}) or without ELEC ({square}) under different osmolar, temperature and pH conditions
Values are means ± S.D.; n = 6.

 
Maximum autolysis was observed when the cells were harvested in the exponentional phase and then incubated in PBS. It was found that P. pentosaceus 16:1 underwent autolysis at pH 6.8, which is within the optimal pH range for LAB lysis, as has been reported previously (pH 6.2–6.8; Mora et al. 2003).

After 72 h starvation, 30% of the P. pentosaceus 16:1 cells (3.3 x 105 cfu ml–1; data not shown) were lysed, measured as OD at 625 nm, both in the ELEC-stimulated and the unstimulated suspensions (Fig. 4). The curve of autolysis was proportional and slow over 10 h. This is in agreement with earlier observations that bacterial cells undergo autolysis slowly and not completely. Turbidity decreased to a minimum of 30% after 10 h of incubation and then remained constant until the end of the incubation period (72 h). Generally, 30% quantitative lysis of bacteria is recognized as being maximal.



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  		Figure 4.  Kinetics of the autolysis of the P. pentosaceus 16:1 cells treated with ELEC ({blacksquare}) or untreated ({square})
Values are means ± S.D.; n = 6.

 
Pure muramic acid (peptidoglycan) of P. pentosaceus 16:1, as a substrate for autolysins, was applied on SDS-PAGE gels (2-DE). The amount of this macromolecule to be used in these experiments had been determined by using a methyl ester O-methyl acetate derivative (GC-MSMS) (Fig. 5).



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  		Figure 5.  Chromatographic profile of muramic acid derivatives
Methyl ester O-methyl acetate muramic acid derivative of P. pentosaceus (A) and spectrum obtained from GC-MSMS (B). kCount, relative amount of the muramic acid.

 
Effect of ELEC on the autolysis of P. pentosaceus 16:1

Stimulation by the ELEC caused qualitative changes in the autolysin production, as shown by comparison with the autolysis present in untreated samples of P. pentosaceus 16:1 after incubation. The pI of the proteins varied between 7.86 and 8.7 (8.03 ± 0.69, n = 12) with relative molecular masses (Mr) ranging from 28 to 230 kDa (n = 12). A representative example of the autolysin profile obtained is presented in Fig. 6. The molecular mass of autolysins ranged from 28 to 230 kDa when the samples were treated with the ELEC (Fig. 6B and Table 1). The profile of autolysins from the control, ELEC-untreated samples had molecular masses ranging between 60 and 180 kDa (Fig. 6A). The number of autolytic proteins in the supernatant from the ELEC-treated suspensions of P. pentosaceus 16:1 was 36 ± 5 (n = 6) and in untreated suspension it was 14 ± 3 (n = 6, P < 0.001; Table 1).



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  		Figure 6.  Pattern of the supernatant protein bands after 24 h starvation of P. pentosaceus 16:1 resolved by isoelectrofocusing and separated with renaturing 12% SDS-PAGE
The zymograms show autolysis of the bacteria untreated (A) or treated with ELEC stimulation (B). Values of Mr are shown on the left of the figure.

 

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  		Table 1. Example of the characteristics of the autolysins (as protein spots) in supernatants obtained from P. pentosaceus 16:1 suspensions treated with electrical current (+ELEC) and untreated (–ELEC)
 
The activity spectrum of autolysins from untreated P. pentosaceus 16:1 varied according to the starvation conditions. It was also possible to identify enzymes with a low Mr (under 45 kDa). These unique hydrolases were detected in a region between Mr 41, 44 and 50 kDa and had a different pI. In these studies, the major autolysin spots were noted in the region of 110 and 170 kDa (Fig. 6B). The autolysis profile obtained was characteristic and similar to that presented by Mora et al. (2003) for P. acidilactici, which showed autolytic activity of molecules ranging between 45 and 110 kDa. It should be mentioned that technically, the process of autolysis and the presence of metal ions in the buffer of bacterial cells was crucial for the zymogram visualization (Strating & Clarke, 2001).

Effect of peptidoglycan hydrolases from ELEC-treated P. pentosaceus 16:1 on LAB strains

The peptidoglycans isolated from the different LAB strains used in this study were submitted to the action of lysozyme, as a control (Table 2), and their stable and identical sensitivity to the action of this enzyme indicated that, although the tested cell walls were acetylated, there was still a substantial effect of the autolysin. It has generally been considered that acetylation of the peptidoglycans would essentially limit their degradation by autolysins, and the degree of peptidoglycan O-acetylation corresponded to the degree of resistance to lysozyme hydrolysis (Clarke, 1993). Thus, acetylation might block the action of specific groups of autolysins while not affecting others. The physiological role of peptidoglycan O-acetylation is not understood, but it might belong to a control system of autolysin action.


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  		Table 2. The effect of P. pentosaceus 16:1 autolysins expressed as the diameter of the transparent zones (mean ± S.D, n = 6) obtained before and after ELEC treatment on the degradation of the host and different species of LAB cell walls
 
The degradation of the peptidoglycans obtained from the different bacteria by the cell-free supernatant of 72 h-starved P. pentosaceus 16:1 bacteria treated and untreated with the ELEC was significantly different P < 0.05 (Table 2). An example is shown of the lysis, caused by the autolysins produced by the ELEC-treated and untreated P. pentosaceus 16:1, of the peptydiglycans suspended in agar gels and isolated from P. pentosaceus 16:1 (Fig. 7A) and L. plantarum 2592 (Fig. 7B).



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  		Figure 7.  Example of the lysis of the peptydiglycans suspended in agar gels and isolated from P. pentosaceus 16:1 (A) and L. plantarum 2592 (B) as a result of the action of the peptidoglycan hydrolases (autolysins) produced by P. pentosaceus 16:1
The extent of the hydrolase activity is indicated by the diameter of the transparency around the application point: 1, supernatant from P. pentosaceus 16:1 treated with ELEC during starvation stress; 2, supernatant from P. pentosaceus 16:1 untreated with ELEC during starvation stress; 3, lysozyme as positive control; and 4, supernatant from P.pentosaceus 16:1 culture before exposure to starvation stress.

 
Similarly to lysozyme, the supernatant from the starved pediococcal suspension treated with ELEC degraded peptidoglycans both from its own cell walls (P. pentosaceus 16:1) and from those of the other bacterial strains (L. plantarum 2592, L. paracasei ssp. paracasei F19, L. mesenteroides 77:1 and the control bacteria M. lysodeikticus ATCC 4698). In contrast, the supernatant from starved pedicocci (P. pentosaceus 16:1) untreated with ELEC acted exclusively against the host peptidoglycan (P. pentosaceus 16:1). As noted above, the ELEC-treated P. pentosaceus 16:1 produced both low- and high-molecular autolysins, while the untreated P. pentosaceus 16:1 produced exclusively the high-molecular weight ones.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Generally considered as safe (GRAS) pediococci can be isolated from meat and gut digesta, fermented vegetables and dairy products, and they are commonly used as probiotics. As for other Gram-positive bacteria, the cell wall of the pediococci is composed of peptidoglycan, with a matrix consisting of polysaccharides, teichoic acids and proteins (Govindasamy-Lucey et al. 2000; Konings et al. 2000). Peptidoglycan (muramic acid), the substrate used in the present studies, is a polymer of amino sugars cross-linked by short peptides which forms a covalent matrix that surrounds the cytoplasmic membrane and constitutes the major skeletal component of the Gram-positive bacteria cell wall. Cell-wall peptidoglycan is strong and highly dynamic; the structure expands as the cell grows and is reshaped when it divides or differentiates. Autolysins are involved in the selective hydrolysis of peptidoglycans and thus can contribute to cellular processes, including proliferation and death (Shandholm & Sarimo, 1981; Tomasz, 1984).

The absolute numbers of autolysins counted on the gels probably did not reflect the true intracellular autolysin profile. It should be noted that about 70% of the bacterial cells which survived the stress of 72 h incubation (two periods of starvation, and treatment with the ELEC) still had their autolysins localized intracellulary. In addition, during bacterial growth, it has been observed that peptidoglycan hydrolases can be processed during their transport through the cytoplasmic membrane, and multiple forms of the same active autolysin can be generated (Leclerc & Asselin, 1989). Thus, it could not be excluded that the ELEC not only affected autolysin production (i.e. number and type), but also may have participated in autolysin remodelling during the transport of these enzymes through the bacterial walls.

The results obtained clearly showed that the ELEC, having characteristics similar to those currents produced by the intestinal wall, qualitatively affected the autolysin production by P. pentosaceus 16:1. In addition, it could be speculated that that the profile of the liberated autolysins depended on the susceptibility of the bacteria to the ELEC under the present experimental conditions. ELEC treatment in vitro allowed for the further identification of high- and low-autolytic P. pentosaceus 16:1 cells. Finally, the profile of autolysins liberated from the cells was associated with the action of ELEC. Considering that autolysins are intensively produced where bacterial cells are actively growing and dividing, it was concluded that the ELEC can regulate this process.

However, it was not possible to define the mode of action of the ELEC. Several questions could be addressed in this respect, e.g. is it purely an electrical effect or is the effect related to the slow electrolysis in the medium caused by the ELEC? Another important question is whether direct or alternating currents of comparable amplitude to those employed in the present series of experiments could produce similar changes in autolysin production. Unfortunately, we were not able to find relevant references in the scientific literature to discuss this intriguing suggestion.

The lack of answers to these questions cannot abolish the importance of the fact that the ELEC, having parameters related to the MMC, can affect bacterial autolysin production. Thus, it is likely that in vivo electrical currents related to the MMC can qualitatively affect the autolysin production by the probiotic bacteria in the gut, and in this way influence the process of natural colonization of the intestine by LAB and other bacteria. Moreover, the clinical relevance to human medicine of this observation should also be addressed, since there are many studies recommending the use of LAB bacteria in the oral treatment of a long list of diseases.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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   Acknowledgements
 
The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS); Swedish Institute (SI), Wisby program; VINNOVA (Eureka, Project nos 2676 and 2675); PANAD (EC project no. QLK2-CT-2000–00411); H. and J. Forssmans Foundation and MIR T-213 research grants are gratefully acknowledged for their financial support.




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