Cell nanomotion (Venturelli et al., 2020) is a significant discovery, which could potentially lead to numerous biological and medical applications. Several scientific groups actively promote the atomic force microscope (AFM)-based method to detect bacterial nanomotion to determine the sensitivity and/or resistance of bacteria to antibiotics: the end of the nanomotion indicates the death of bacteria and the effectiveness of the drug (Longo et al., 2013; Kasas et al., 2015). This allows getting the most important clinical data on antibiotic resistance in minutes as opposed to several days required in the case of the classic disk diffusion method. It is especially important to provide timely results if the growth of bacteria is very slow (for example, in bacteria with an acid-resistant cell wall), which requires weeks to give a recommendation on antibiotic, while the use of a sensor that detects nanomotion makes it possible to determine antibiotic resistance in Mycobacterium in a few hours (Mustazzolu et al., 2019). New systems based on the combination of piezoelectric elements, microfluidics, and optics allow detecting the sensitivity and/or resistance of bacteria to antibiotics in 7–15 min (Johnson et al., 2017). The use of a cantilever functionalized with antibodies can significantly reduce the time of specific diagnostics of the pathogen, since a unique immune complex is formed. At the same time, the sensitivity of the method turns out to be very high: a few hundred bacteria adhered to the cantilever are enough to obtain a reliable and reproducible signal, and this number can even drop to one single cell in the case of larger microorganisms such as yeast (Kasas et al., 2021). Thus, using only the oscillations of the AFM cantilever, it is possible to carry out a full cycle of express diagnostics includes both determining the type of microorganism and antibiotic resistance.

In general, nanomotion characterizes the metabolic activity of bacteria and can be considered as a kind of “signature of life” (Kasas et al., 2015). The binary answer of the system, i.e., presence of “nanomotion equals life” and “no nanomotion signifying death” is very convenient and seems universal. So, just as in the case of bacteria, nanomotion detection can be used to select antitumor therapy, when cancer cells are fixed on the cantilever and effective therapeutic agents are selected according to the change in the cantilever oscillations (Wu et al., 2016; Stupar et al., 2021).

In 2021, it was found that the AFM oscillation is so sensitive and specific that it can even be used to distinguish virulent from avirulent Bordetella pertussis cells (Villalba et al., 2021). Authors used the same virulent and avirulent B. pertussis Tohama I strain (avirulence was modulated by medium) with same characteristics (mass, metabolic activity, and morphology) and received different oscillation patterns depended on virulence. Thus, it is presumed that strains with more distinctions in biochemical properties, mobility and mass should have even much expressed differences in nanomotion.

In this work, we aimed (1) to compare the features of nanomotion in bacteria of different species with different biochemical profiles; (2) to detect a phagocytosis of bacteria by neutrophil granulocytes (NGs) using AFM-based nanomotion detection.

For these experiments, we analyzed different clinical bacterial isolates obtained from different patients with different pathologies. Only two biochemical traits of the 28 studied ones were common to all isolated strains: all of them fermented glucose and did not dehydrogenate arginine. Otherwise, the biochemical profiles of the studied bacteria were significantly different. Some significant differences were also noted in the morphology and ultrastructure of bacteria (Figure 2). The profile of bacterial nanomotion studied by AFM-based nanomotion detection also showed that each strain has its own ‘signature’ which was revealed by the change in the DFL signal (Figure 3A). Interestingly, the mean values of the DFL signal did not show statistically significant differences (p > 0.05, Figure 3C), so this criterion cannot be used to describe the features of nanomotion of different strains. On the contrary, variance seems to be a criterion that maximally reflects the features of bacterial nanomotion. The analysis of the distribution of variances (Figure 3C) showed that the maximum changes in the DFL signal have been noted in E. coli 321 and P. mirabilis 649–2. These strains have peritrichous flagellation, which provides them with maximum mobility under physiological conditions and at the same time constitutes one of the pathogenicity factors (Manos et al., 2004; Grognot and Taute, 2021). It can be concluded that the presence of flagella makes a certain contribution to bacterial nanomotion. However, the change in the DFL cannot be explained only by the presence of flagella, because in the other two studies, strains of S. aureus 2879 M and K. pneumonia 173-P2 also exhibit nanomotion. Although these changes were smaller compare to experiments with strains with greater activity, they were statistically significantly larger (p < 0.01) than the control signal obtained from the oscillation of an empty functionalized cantilever.

To study the functional activity of NGs, cells were attached to the cantilever surface. Since NGs have a wide range of adhesins, they are able to independently firmly bind to any substrate (Pleskova et al., 2018b). However, in this series of experiments, weak fixation with 0.01% poly-L-lysine was used. The nanomotion signal of unprimed NGs was indistinguishable from the control (results not shown). Before a significant nanomotion for different types of mammalian cells in many articles was shown such as cancer cells line MCF-7 (Wu et al., 2016), sperm cells (Wu et al., 2017), and osteoblasts and neurons (Kasas et al., 2015; Kohler et al., 2019). Also the mass of mouse fibroblasts and HeLa cells was assessed (Martínez-Martín et al., 2017). However, there were either malignantly transformed cells (HeLa, MCF-7) or osteoblasts which have high metabolic activity, or cells with high mobility (sperm cells). Whereas, NGs are a special type of cells and the absence of basic activity can be explained by the features of the neutrophil life cycle. Unprimed neutrophil has a low metabolic activity level (for example, even for the synthesis of ATP molecules it uses glycolysis but not oxidative phosphorylation (Mazaki et al., 2019)) and by this the absence of the initial nanomotion of NGs is explained. After the activation of neutrophil metabolic activity of the cell rises significantly, in particular, a respiratory burst with ROS production is generated in cells, cell migration activity and biosynthesis processes are activated, and nanomotions appear. Theoretically, the adhesion process could change the basic level of NGs priming (Vogt et al., 2018), but in our experiments, it did not lead to any detectable oscillations. In order to further study the functional activity of NGs, we induced phagocytosis, which is always accompanied by a powerful functional rearrangement of cells, such as: the assembly of the NADPH oxidase complex, the release of ROS and reactive nitrogen species, the formation of hypochloric acid and hypochlorite, the activation of all intracellular enzymes, and the formation of cytokines. It can advance so powerfully that in some cases, it leads to cell death (Pleskova and Mikheeva, 2018). Therefore, given the multi-stage nature of phagocytosis, more research is needed in order to determine which stages of the process have the greatest impact on the cantilever oscillation. It must be remembered that the phagocyte, in addition to the respiratory burst, can form pseudopodia and can migrate in the direction of the chemoattractant agents. All these factors can make a significant contribution to the cantilever oscillations. Since the release of ROS strongly correlates with the activation of cells, we first determined which of the strains would cause the maximum release of ROS.

Using the method of luminol-dependent chemiluminescence, it was shown that the S. aureus 2879 M induces maximum production of ROS by NGs, which in 20–30 min caused the release of ROS several times higher than in the case of other strains (Figure 4). In our previous studies it was also shown that NGs absorb S. aureus more actively than E. coli, which was determined by the phagocytic number and phagocytic index (for S. aureus, 50% phagocytosis was observed after 7 min, and for E. coli after 11 min) (Pleskova et al., 2018c).

The incubation of NGs with S. aureus 2879 M showed a statistically significant increase in the signal 15 min after co-incubation with the bacteria. The cellular nanomotion also persisted after 30 min co-incubation (Figure 5). These results do not contradict the results of ROS formation, since Figure 4 shows that by 15 min, the respiratory burst increases exponentially, and by 30 min, it is at the maximum level. Most likely, the formation of pseudopodia (Figure 6) and, possibly, limited migration of NG can also contribute to nanomotion. The possibility of the migration process is indirectly evidenced by the presence of cellular “shadows” on the cantilever at the sites of primary cell adhesion.

The implementation of the process of phagocytosis is also evidenced by Figure 6, on which bacteria inside NG are well defined. One of the NGs obviously died as a result of the phagocytosis process.

Nanomotion registered using AFM cantilever seems to be a universal marker of the activity of both prokaryotic and eukaryotic cells. All bacteria, regardless of the source of isolation, features of morphology, ultrastructure, and biochemical profile, caused significant changes in the DFL signal compared to the control (an empty functionalized cantilever in the same nutrient medium). We found out that the variance values are the best metric to monitor cellular nanomotion. Nanomotion was more pronounced in flagellar forms of bacteria, although the cantilever oscillation cannot be explained by the presence of flagella alone, since metabolic processes are actively running in all bacteria, which allows bacterial cells to divide.

For NGs adhered to the surface of the cantilever in the unprimed state, no change in the analytical signal was observed. However, the implementation of NGs physiological functions such as phagocytosis, which is accompanied by cell priming and rearrangement of the metabolic activity, caused an increase in cantilever oscillation, which was observed after a 15 min co-incubation with bacteria and remained high for up to 30 min. Thus, nanomotion directly reflects the metabolically active state of cells.

A graphical abstract of study showing the affection of different bacterial and eukaryotic cells factors on the oscillations of cantilever is presented in Supplementary Figure S3.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Bioethics Commission of Lobachevsky State University (created on November 11, 2016, order on creation No. 497-OD). The patients/participants provided their written informed consent to participate in this study.

SP and RK: conceptualization. EL: experiments with bacteria. SB and EL: experiment with neutrophil granulocytes. AB: experiments with electron microscopy. EL, RK, and SP: statistical analysis. SP: project administration. SP and NB: writing the article. NB: translation and correction. All authors have read and agreed to the published version of the manuscript.

The work has been performed in the frame of the program of scientific academic leadership ‘Priority-2030’ of Lobachevsky State University of Nizhny Novgorod and had financial support only by Russian Science Foundation, project No. 22–14-20001.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.