Microbial Pathogenesis 109 (2017) 4e7

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Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Enhancing effect of 50 Hz rotating magnetic field on induction of Shiga

toxin-converting lambdoid prophages
M. Struk a, B. Grygorcewicz a, P. Nawrotek a, *, A. Augustyniak a, M. Konopacki b,
M. Kordas b, R. Rakoczy b
a
Department of Immunology, Microbiology and Physiological Chemistry, Faculty of Biotechnology and Animal Husbandry, West Pomeranian University of

w 45, 70-311 Szczecin, Poland
Technology, Szczecin, al. Piasto
b
Department of Chemical Engineering, Institute of Chemical Engineering and Environmental Protection Processes, West Pomeranian University of

w 42, 71-065 Szczecin, Poland
Technology, Szczecin, al. Piasto

a r t i c l e i n f o a b s t r a c t

Article history: Studies aimed at investigating factors and mechanism of induction of prophages, a major pathogenesis
Received 2 September 2016 factor of Shiga toxin-producing Escherichia coli (STEC), are considered important to develop an effective
Received in revised form treatment for STEC infections. In this study, we demonstrated the synergistic effect of the rotating
8 May 2017
magnetic field (RMF) of induction B ¼ 34 mT and frequency G ¼ 50 Hz at a constant temperature of 37  C
Accepted 11 May 2017
and mitomycin C (MMC), that resulted in a higher level of induction of stx-carrying lambdoid Stx pro-
Available online 12 May 2017
phages. This is a first report on the induction of lambdoid Stx prophages in response to the enhancing
effect of popular inductor (mitomycin C) under the influence of RMF.
Keywords:
Rotating magnetic field
© 2017 Elsevier Ltd. All rights reserved.
Mitomycin C
STEC
Induction
Stx prophages

1. Introduction
The Shiga toxin (Stx) is a major pathogenesis factor of Shiga
toxin-producing Escherichia coli (STEC). The Stx genes (stx1, stx2)
are located in genomes of Shiga toxin converting lambdoid pro-
phages (also referred to as Stx prophages), which are mobile ge-
netic elements [1]. Expression of the stx gene is under control of the
phage's late genes promoter. According to this phenomenon, the
synthesis of the Shiga toxin is normally repressed, and occurs only
after induction of Stx prophages by various biological, chemical,
and physical factors. A special role is played by the factors
damaging genetic material, of which UV radiation and mitomycin C
(MMC) are best known [2]. Antibiotics are also considered the
inductive factors and for that reason STEC infections cannot be
treated with these substances [3].
STEC strains do not have systems of the Shiga toxin secretion,
therefore the release of toxins is subsequent to lysis of the cells and
the release of progeny phages [4,5]. The significant increase of Shiga
toxin production during the induction of prophages suggests that it
* Corresponding author.
E-mail address: pawel.nawrotek@zut.edu.pl (P. Nawrotek).
may play a direct and important role in the modulation of the
expression of STEC virulence factors encoded in the genomes of
lambdoid phages [5].
The effective and specific treatment of STEC-caused infections
has not been developed to date [3]. Therefore, understanding the
role of various factors causing the stx prophages induction is
essential for developing a treatment which could eliminate enter-
ohemorrhagic E. coli without inducing the Stx prophage.
Numerous experimental investigations upon organisms have
been carried out with an effort to detect the biological effects of
static magnetic fields (SMFs). These studies cover a wide range of
topics, including both biological systems and magnetic fields
strength. For example, strong SMFs (typically greater than 0.2 T)
found versatile applications in biological systems: genotoxic effect
and mutation studies [6]; chromosomal damage [7]; DNA damage
[8]; proliferation [9]; gene expression [10,11]; metabolic activity
[12]; aggregation and cell adhesion [13]. Nevertheless, there are
only few studies regarding the effect of rotating magnetic field
(RMF) on microorganisms where the increase of functional pa-
rameters was observed (e.g. cell metabolism or proliferation pa-
rameters) [14e16]. The RMF is a magnetic field that changes
direction (ideally) at a constant angular rate. This is a key principle

http://dx.doi.org/10.1016/j.micpath.2017.05.018
0882-4010/© 2017 Elsevier Ltd. All rights reserved.
 M. Struk et al. / Microbial Pathogenesis 109 (2017) 4e7
in the operation of the alternating-current motor. RMFs are mainly
utilized in electric rotating machinery e.g. electric generators or
induction motors, and therefore electromechanical applications.
Furthermore, the RMF arises as a resultant field during the super-
position of two or more alternating current magnetic fields
(ACMFs) of identical frequency but spatially displaced in phase with
respect to one another. This kind of MF is used in electrical motors,
measuring instruments, and various AC regulation and control
equipment [17]. The knowledge regarding the influence of RMF on
cellular processes is still insufficient, and data on its role in the
mechanism of the prophages induction still lacks.
Therefore, the aim of this study was to analyse the effect of in-
duction of stx-carrying lambdoid Stx prophages from Escherichia
coli by 1-h exposure to RMF (B ¼ 34 mT, f ¼ 50 Hz) and popular
inductor e mitomycin C (0.5 mg  mL1).
2. Materials and methods
2.1. Exposure to RMF and mitomycin C
The exposure of Stx prophages-carrying bacterial cells to RMF
and mitomycin C (Sigma-Aldrich, USA) was conducted in the
experimental set-up consisting of the RMF generators described by
Nawrotek et al. [15].
2.2. The biological material
Prior to the study, two E. coli strains: MG1655 lysogenic with
bacteriophage 933 W Dstx2:catGFP [18], courtesy of Department of

sk, Gdan

sk, Poland, and a
Molecular Biology, University of Gdan
wild-isolate labelled W10 (sheep origin, carrying stx1 and stx2
genes), deposited in Department of Immunology, Microbiology and
Physiological Chemistry, were used. For phage titration, E. coli
MG1655 was used as a host.
2.3. Optical density (OD) measurements
The optical density of the bacterial cultures was measured for a
purpose of phage titration every 30 min at the wavelength of
600 nm in 96 well microtiter plates with 100 mL of each sample
using Infinite 200 PRO NanoQuant microplate reader (Tecan,
Ma€nnedorf, Switzerland). The measurements were carried out in
three replicates.
2.4. Monitoring the prophage induction
Bacterial strains were cultured overnight in LB medium (Luria-
Bretani, Oxoid, UK) at 37  C with shaking (180 rpm). Afterwards, the
fresh medium was inoculated at a 1:100 ratio with overnight cul-
tures and incubated with shaking at 37  C to obtain optical density
OD ¼ 0.1 at 600 nm. Prepared cultures were vortexed and
dispensed in the volume of 10 mL into falcon tubes (15 mL) into
four parts. Two inductive agents were used in three experimental
setups for each individual strain: RMF (50 Hz, one hour), mitomycin
C (0.5 mg  mL1) and both agents together. The control was
cultured without any inductive agent. Samples, that were not
exposed to the RMF, were incubated for one hour without shaking
at 37  C. Eventually, the culture was continued at 37  C with
shaking for 7 h.
Samples were collected every 30 min. Phage lysates were pre-
pared. For this purpose, aliquots of 300 mL of the induced bacterial
culture were vortexed with 30 mL of chloroform, then centrifuged.
Lysates were titrated using double layer agar method. Top-agar
medium, preheated to 45  C, was mixed with overnight culture of
MG1655 and suspensions from serial dilutions of phage lysates in
5
TM buffer (Tris-HCl, pH 7.2; MgSO4 10 mM). The bottom layer was
previously supplemented with chloramphenicol (2.5 mg  mL1,
Sigma-Aldrich, USA) [19]. Plates were incubated at 37  C for 24 h.
The phage titre was calculated based on the number of plaques and
described as plaque-forming units (PFU). The experiment was
conducted in triplicate.
2.5. Analysis of mitomycin C UV-VIS spectra
UVeVIS spectra of MMC aqueous solution have been tested to
exclude the impact of RMF on cytostatic structure. Therefore,
UVeVIS spectra of mitomycin C aqueous solution were recorded
with computer-controlled spectrophotometer V-670 Jasco (Jasco
Inc., USA), within the range of 235e600 nm at 25 ± 0.1  C.
2.6. Statistical analysis
For statistical analysis of the results, the t-test was performed. P
values of <0.05 were considered significant. All statistical analyses
were conducted with GraphPad Prism 5.02 (La Jolla, CA, USA)
software.
3. Results
3.1. OD of bacterial cultures
Exposure of analysed E. coli strains (model and wild isolate) to
the RMF of induction B ¼ 30 mT, frequency 50 Hz and 1-h exposure
caused no significant difference in comparison to the ODs of the
control and treated samples. The optical density of model strain
simultaneously treated with mitomycin C (0.5 mg  mL1) and RMF
in defined parameters decreased significantly (p < 0.05) in relation
to the samples treated only with mitomycin C. There were no sta-
tistically significant changes (p ¼ 0.1514) of OD values for wild
isolate in samples treated only with mitomycin C as compared to
those treated using both factors. The growth curves are presented
in Fig. 1.
3.2. Prophage induction
The cultures of both E. coli strains were exposed to mitomycin C
in LB broth at 37  C, whereas the influence of RMF on phage in-
duction in strains was conducted in field generator. Fig. 2 (A and B)
shows that the cooperation of mitomycin C and RMF resulted in
higher relative phage titre. The phage titre of the reference 933 W
phage in response to mitomycin C together with the exposure to
RMF after 270 min was 3.37  107 PFU  mL1, which was signif-
icantly higher than in the control sample induced only with mito-
mycin C, for which the titre counted as 1.1  107 PFU  mL1. Wild
type stx-converting phage from W10 strain was also induced after
mitomycin C treatment. Relative titre of this phage after exposition
to mitomycin C for 120 min was 2.46  105 and increased to
1.3  106 PFU  mL1 once cultures were exposed to RMF. However,
after this time, in the case of bacteriophage W10, a rapid decline in
relative phage titre to 6.5  105 PFU  mL1 was observed, due to
the adsorption of released progeny bacteriophages to their host cell
(Fig. 2B).
3.3. UV-VIS analysis of mitomycin C
UV-VIS spectroscopy showed the characteristic strong absorp-
tion of mitomycin C at 363 nm (Fig. 3). After exposure to the RMF
(50 Hz, 1 h), the detected absorption was consistent with the con-
trol not exposed to MMC.
6 M. Struk et al. / Microbial Pathogenesis 109 (2017) 4e7

Fig. 1. Culture density (by measurement of A600 nm, OD600 nm) of the tested stains (wild isolate e A and the model strain e B). Experiments were repeated in triplicate with a high
reproducibility (SD < 20%). Representative results are shown.

Fig. 2. Efficiency of prophage induction. The relative phage titre was calculated by subtraction of the phage titre determined in an uninduced culture from the titre determined in
the induced culture. A e relative phage titre of bacteriophage 933 W; B e relative phage titre of bacteriophage from the wild strain (W10). Experiments were repeated in triplicate
with a high reproducibility (SD < 20%), and representative results are shown.

4. Discussion
Induction of Shiga toxin-converting prophage and expression of
stx genes correlated with this phenomenon is essential for STEC to
attain maximal pathogenicity. The induction may occur spontane-
ously under the influence of various external (environmental)
factors such as antibiotics (e.g. norfloxacin), cytostatic (e.g. mito-
mycin C) or hydrogen peroxide [2,4]. In this study, we evaluated
whether the RMF of induction B ¼ 34 mT and frequency G ¼ 50 Hz
at a constant temperature of 37  C has an effect on the induction of
lambdoid Stx prophages from cells of Shiga toxin-producing E. coli.
Our study shows the synergistic effect occurring between mito-
mycin C and RMF of specified parameters.
To our knowledge, it is the first report on the induction of Stx
prophages in response to the synergistic effect of popular inductor
(MMC) and RMF. It should be also underlined that exposure to RMF
did not alter the UV-VIS spectra of MMC (Fig. 3), which indicates
that the drug retained its structure during treatment. Referring to
Hadas et al. [20], who indicated that E. coli cells that are multiplied
and metabolized faster, are larger and have their translation ma-
chinery up-regulated. Consequently, this causes a rise in number of
produced virions leading to a larger burst size. Nejman et al. [21]
suggested that effectiveness of intracellular processes after the
induction of prophage (e.g. phage DNA replication), are crucial for
the development of bacteriophage. Additionally, Nawrotek et al.
[15] reported the effect of the growth and metabolic activity
stimulation in E. coli that was caused by 50 Hz RMF. Hypothetically,
the rotating magnetic field amplifies the induction rate of lambdoid
prophage caused by other factors like mitomycin C, by the influence
on cells proliferation and their metabolic activity, which as a result
significantly increases the synthesis of progeny viral particles.
Furthermore, we assume that better understanding of the
 M. Struk et al. / Microbial Pathogenesis 109 (2017) 4e7
Fig. 3. UV-VIS absorption spectra of mitomycin C exposed and unexposed to RMF.
mechanism and factors causing higher induction level could also
set a ground for novel methods applicable in the production of
bacteriophages that are commonly used in biotechnology.
Stx prophages are involved in spreading and survival of patho-
genesis factors (stx1 and stx2) in the natural environment. Induc-
tion of these viruses plays a role in spreading of Shiga toxin genes
via horizontal gene transfer to non-pathogenic E. coli, which can
pose an epidemiological hazard. As mentioned previously, many
factors can influence on the induction of prophages including an-
tibiotics, UV, hydrostatic pressure, temperature or H2O2 that are
well known [2,22]. The prophage induction by mitomycin C de-
creases the OD of bacterial culture due to the lysis of cells, and is
associated with lytic development of phages and the release of
progeny virions. Statistically, culture OD between the MMC-treated
and treated with MMC in conjunction with the RMF was signifi-
cantly different (p < 0.05). This may be related to the increasing
level of the prophage induction and stronger cell lysis, induced by
the enzymes of progeny phages, when both factors were used
together. The OD values measured in the case of the wild isolate
(W10) carrying stx1 and stx2 genes were similar to OD values after
exposure to MMC and both RMF with MMC, which may indicate a
high level of prophage induction. These results may also demon-
strate a relationship between the induction of stx-carrying lamb-
doid prophages depending on the integration locus, and the
quantity and ability of phages to remain stable at the host genome
[4]. Ło
s and We˛grzyn [4] reported that when the two prophages
encoding the Shiga toxin in bacterial genome are integrated after
treatment with the inducing agent, only one of them induces.
Our results indicate that RMF may be considered an enhancer to
other factors. Both analysed E. coli strains (model and wild isolate)
confirm increasing induction level of the prophage after exposure
to MMC and RMF together. Although bacteriophage from the wild
strain (W10) was induced by described factors, this effect was
shorter in comparison to bacteriophage 933 W.
7
Declaration of interest
The authors report no conflicts of interest.
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