Adhesion of gram‐negative rod‐shaped bacteria on 1D nano‐ripple glass pattern in weak magnetic fields

Abstract This research project has major applications in the healthcare and biomedical industries. Bacteria reside in human bodies and play an integral role in the mechanism of life. However, their excessive growth or the invasion of similar agents can be dangerous and may cause fatal or incurable diseases. On the other hand, increased exposure to electromagnetic radiation and its impact on health and safety is a common concern to medical science. Some nanostructure materials have interesting properties regarding facilitating or impeding cell growth. An understanding of these phenomena can be utilized to establish the optimum benefit of these structures in healthcare and medical research. We focus on the commonly found rod‐shaped, gram‐negative bacteria and their orientation and community development on the cellular level in the presence of weak magnetic fields on one dimensional nano‐ripple glass patterns to investigate the impact of nanostructures on the growth pattern of bacteria. The change in bacterial behavior on nanostructures and the impact of magnetic fields will open up new venues in the utilization of nanostructures. It is noticed that bacterial entrapment in nano‐grooves leads to the growth of larger colonies on the nanostructures, whereas magnetic fields reduce the size of colonies and suppress their growth.

microspheres of silver zinc oxide resist the growth of bacteria and biofilms (Patil et al., 2014). The arrangement of bacterial colonies and formation of biofilms on nanopatterns facilitates metabolic reactions (Hochbaum & Aizenberg, 2010). Thus, it is essential to understand chemical and physical interactions between bacteria and nanosurfaces.
On the other hand, the nature of bacteria is very adaptive and how it survives under different conditions of density, temperature, pressure and magnetic field is of tremendous importance in medical and clinical science. Microorganisms have adapted to fluctuations in the Earth's magnetic field over a period of time. However, the response of bacteria to other external magnetic field influences is still being investigated (Brkovic, Postic, & Ilic, 2015;Kohno, Yamazaki, Kimura, & Wada, 2000). In space, humans encounter a different environment of radiation and electromagnetic forces. One interest these days is to inspect the magnetic field influence on human bodies in outer space, outside of the influence of Earth's gravity and its magnetic field. Electromagnetism has significant importance in understanding living organisms. Electromagnetic signals are received from the brain, heart, and isolated nerves, and signals are also emitted during the process of muscle building. Bacteria grow through the process of binary fission, and microbial growth strongly depends on factors such as ion transportation through the cell walls, their cellular structure, atomic and molecular interactions, enzymatic activities, other protein functions and the neural network in the living organisms.
All these processes are facilitated by electromagnetic interactions.
In some animals, bacteria consist of magnetic particles, mainly connected to the neural tissues, which determine the direction of their movement. The rapid increase in the use of magnetic field devices for medical use such as in radiology, cardiology, dentistry, oncology, and neurosurgery (Mornet, Vasseur, Grasseta, & Duguet, 2004) has made it important for us to understand the effect of magnetic fields on living organisms in the use of biomedical applications.
Strong magnetic fields can have strong effects (Hardell & Sage, 2008;Lai & Singh, 2004), such as the breaking and forming of chemical bonds which play a big role in the synthesis of DNA and thus cellular growth. However, weak magnetic field effects are not negligible. Although it may be challenging to design and conduct such experiments, they are of significant importance, as interaction with magnetic devices is unavoidable and continuous perturbation may result in permanent changes. Hence, the study of biological effects of magnetic fields is very crucial to understand microbial growth.
The perturbative effects caused by weak magnetic fields have a huge impact on some organisms (Frankel, 1979;Masood, 2017).
Since bacteria are single-celled organisms, it is easy to study the effect of magnetic fields on their growth and cell structure, which may in turn be used to control and treat microbial diseases. This approach can later be extended to get detailed information about cellular structures.
The aim of this paper was to study the weak magnetic field influence on the growth of E. coli and Pseudomonas aeruginosa on nanoripple glass structures in comparison with plain glass cover slips.
Some studies show that low frequency alternating magnetic fields tend to slow down the growth rate of E. coli (Aarholt, Flinn, & Smith, 1981;Cellini, 2008;Del Re, Bersani, Agostini, Mesirca, & Giorgi, 2004;Justo, Pérez, Alvarez, & Alegre, 2006;Segatore et al., 2012;Strasák, Vetterl, & Smarda, 2002). It is interesting to consider the adhesion of E. coli and its growth on nanostructure glass samples. The idea was influenced by the regeneration of the tissues and functional recovery following pathways set by nanostructures such as the spinal nerve cell growth guided by nanotubes or other biomaterial designs (Straley, 2010;Subramanian, Krishnan, & Sethuraman, 2009;Usmani et al., 2016). In this experiment, bacterial adhesion to the surface is enhanced due to the interaction of microorganisms with the nanoripple pattern. The bacterial colonies are bigger on nano-ripple structures, but in the influence of weak magnetic fields, the colonies grow smaller in size. We studied the effect of both homogeneous (uniform electromagnetic field) and nonhomogeneous (nonuniform bar magnetic field) magnetic fields on the formation of bacterial colonies on nano-ripple structures and plain glass cover slips.

| Fabrication of glass nano-ripple
We engineer a nano-ripple pattern on glass cover slips by means of oblique angle gas cluster ion beam (GCIB) irradiation (Kirkpatrick, 2003;Yamada, 1995;Yamada, Matsuob, Toyodaa, & Kirkpatrickc, 2001) (see Figure 1) using an Epion cluster ion implanter. Clusters of argon gas (each with about 3000 atoms of argon) of energy 30 keV bombard the glass surface at an incident angle of 60°. The off-normal cluster ions and the surface atoms undergo synergistic interactions which cause the forward sputtering of atoms. As a result of forward sputtering and surface diffusion of the glass molecules, a nano-ripple pattern is obtained (Lozano et al., 2013;Tilakaratne, 2012;Toyoda, Mashita, & Yamada, 2005). The geometry of the nano-ripple structures is tunable by changing the GCIB irradiation fluence . For the cases discussed, we irradiate the glass cover slip with a GCIB fluence of 5 × 10 16 clusters/cm 2 . The cluster ion beam flux is kept constant at approximately 3.9 × 10 12 clusters cm −2 sec −1 .
Cluster formation is a complicated process, and the theoretical descriptions of this process are limited. The phenomena of nucleation and growth take place through the birth of small molecular clusters that form and grow by molecular collisions. Cluster-cluster aggregation becomes more evident when the number of the clusters are large. The detailed mechanism of GCIB induced ripple formation is explained in detail by Tilakaratne et al. in reference . Figure 2 shows an atomic force microscope (AFM) image of the nano-ripple arrays fabricated under selected conditions of 5 × 10 16 clusters/cm 2 GCIB fluence, and 60 degrees GCIB incident angle.

| Experimental procedure
The glass substrates and the plates were thoroughly cleaned and autoclaved in order to remove all impurities and preexisting bacteria.
Cultures of Escherichia coli and Pseudomonas aeruginosa were initially grown in liquid solutions, with each bacteria having a single mother culture. These mother cultures were vortexed, and then quickly pipetted in similar volumes onto multiple cleaned glass nano-ripple structures, where they remained for 3 days at room temperature.
We had three plates for each bacterial species growing on a plain glass for comparison. After sufficient growth, we washed the substrates with phosphate-buffered saline (PBS) to remove the unattached bacteria from the surface. The bacteria was then stained using a gram crystal violet solution. The glass substrates were washed once again with the buffer solution to get rid of excess dye.
The images of the bacteria on plain and nano-ripple glass were taken under a microscope of 100× magnification.
Bacterial growth was studied at room temperature in two different types of weak magnetic fields. One set of E. coli plates was placed on a collection of permanent weak bar magnets arranged side-by-side as shown in Figure 3a,b, which generates a randomly distributed static weak magnetic field. This magnetic field varies rapidly within short distances, and cannot be represented in a well-defined functional form. However, taking measurements at several nearby points, we noticed that the magnitude is always less than 5 Gauss (0.5 mT). A simple representation of the magnetic field lines due to these alternating magnets can be seen in Figure 4. The other arrangement was the uniform magnetic field generated by several interconnected Helmholtz coils with a weak DC current shown in Figure 3c,d. It is a weak static magnetic field of value 5 Gauss (0.5 mT). The inclusion of both uniform and nonuniform magnetic fields of similar maximum intensity allows us to check for effects which may be caused by the gradient of a magnetic field, rather than simply due to field intensity. For Pseudomonas aeruginosa, a set of plain and nanostructure glass surfaces were placed on the bar magnets. The experiment has been repeated more than three times with similar conditions and new cultures, and the reported results were reproduced.

| RE SULTS AND D ISCUSS I ON S
It is noticed that Escherichia coli shows more growth on glass nanoripple surfaces than on plain glass. The nano-ripple pattern traps the microorganisms in nano-grooves of comparable size due to their alignment along the grooves and causes them to grow into larger colonies. Due to increased bacterial adhesion on the nanopattern, we observed an increase in the number of bacterial colonies compared to the ones on plain glass surfaces. Figure 5 shows microscopic images of Escherichia coli colonies on glass nano-ripple structures in contrast to similar images on plain glass in Figure 6.
The magnification scale is kept the same in Figures 5 and 6 for comparison. The difference in the sizes of colonies is significant on ripple structures ( Figure 5) and the plain glass ( Figure 6). The F I G U R E 1 Schematic diagram of the oblique angle gas cluster ion beam (GCIB) irradiation used in the fabrication of nanoripple glass array structures F I G U R E 2 Atomic force microscope image of the nano-ripple array on the glass surface obtained by GCIB irradiation fluence of 5 × 10 16 clusters/cm 2 , for an incident angle of 60° nano-ripple structures behave as nanochannels for bacteria entrapment. Further investigation will help to develop tools to study the available theoretical models (Odijka, 2006) of cell confinement in nano-grooves.
In addition, the structural effect was studied in the presence of magnetic fields. For this purpose, bacteria was simultaneously grown on nano-ripple glass in uniform and nonuniform magnetic fields along with the plain glass. Figures 7 and 8 compare the bacterial growth on the bar magnets (nonuniform field) and in the uniform magnetic field and show that growth under the two magnetic fields affects the size of the colonies differently.
In the uniform field, the bacterial colonies spread themselves on the surface in a relatively uniform fashion and are small in size. In the nonuniform field, the colonies are relatively larger but still much smaller in comparison to the ones grown without the magnetic field's influence, and the colonies are also not uniformly distributed over the surface.
A pattern in the distribution of bacterial colonies in Figure 7a,c is observed, which may be due to the alignment of bacteria along the magnetic field lines of bar magnets. Conversely, the random distribution of these colonies in Figure 7b could be due to the constructive and destructive interference of the fields from the multiple bar Magnetic field effects on the growth of Pseudomonas aeruginosa reveal similar results (Figure 9), but the colonies of Pseudomonas aeruginosa are larger in size than the Escherichia coli. This may be due to the difference in adhesive properties of the two bacterial species.
In the influence of a magnetic field, bacterial colonies are smaller on glass nano-ripple surfaces ( Figure 10). As seen in Figure 11 terns. If the effect on DNA is proved, it means that the behavior of bacteria will significantly change and their effect on the human body and the response to antibiotics or other drugs needs a detailed investigation. We have focused on the study of the rod-shaped gramnegative bacteria on a comparable size of nano-grooves. However, these effects are expected to be different on different bacterial strains depending on the shape, size, and gram staining properties.

ACK N OWLED G M ENTS
This work was partially funded by the state of Texas through the Texas Center for Superconductivity at the University of Houston.

Partial support was provided by Faculty Research and Development
Funds (FRSF) of University of Houston -Clear Lake (UHCL). Authors would also like to acknowledge the support of the department of Biological Sciences and the chair Dr. Lawrence Rhode.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest associated with the material of the paper.