rus
Issue #1/2022
I.V.Yaminskiy, A.I.Akhmetova
SCANNING PROBE MICROSCOPY OF BACTERIA: GENOTYPE AND PHENOTYPE
SCANNING PROBE MICROSCOPY OF BACTERIA: GENOTYPE AND PHENOTYPE
10.22184/1993-8578.2022.15.1.38.43
INTRODUCTIONS
As an example, let us consider the cases that are quite rare, but which cannot theoretically be omitted. Let us assume that there were only non-viable or dead bacteria in the sample and in the body itself, which may no longer cause harm to the body. A PCR test detects DNA and signals a presence of dangerous pathogens. The doctor prescribes a course of antibiotics as recommended, which, in this case, is useless and can be harmful. Another possibility is that the bacteria are still alive, but they have lost some of their enzyme activity due to the treatment they have already received. Again, the genetic test signals a danger that no longer exists.
Whole DNA can be found in a mammoth that is 5 million years old. The DNA is there, but there is no living mammoth. Perhaps in future scientists will correct this gap. In this example it turns out that the mere presence of DNA in the sample taken does not yet indicate a presence of the carrier itself.
There is a legitimate question. Will all bacteria with the same genotype also have the same phenotype? In other words, are twin bacteria exact copies of each other? Probe microscopy provides an answer to this question. If you take a single bacterium and grow a colony on nutrient medium, it is easy to see that the bacteria in the colony are of different sizes and slightly different shapes (see Fig.1). It often happens that the larger bacteria grow on the edges of the colony. Strictly speaking, no two bacteria are exactly alike in a sea of bacteria. The reverse situation is often found in twins. It is a complete or very close external resemblance.
Which bacteria are potentially dangerous to humans? It is of the kind that scientists have already put on the list of dangerous bacteria. But for a bacterium to become really dangerous, it has to live a full life, including moving and multiplying by fission. For example, every 20–40 minutes. Such pathogenic bacteria pose the greatest danger to the development of disease.
Is it necessary to study the phenotype of bacteria, e.g. appearance, structure, features, etc.? What is more, not a static phenotype, but a phenotype in dynamics, in development. A complete description of a bacterium should include a description of the genotype (genetic protocol) and a description of the phenotype in living. It is the second description that the scanning probe microscopy allows.
RESEARCH METHODS
Since 1996, the probe microscopy has become an important tool for observation of bacterial cells. Even at that time in collaboration with the Gamaleya Institute (Gamaleya Research Center for EMS in the Russian Ministry of Health), it was possible to detect a difference in the parental strain of Escherichia coli and the transductant strain obtained from the parental strain by adding the rfb-a3,4 gene from Shigella flexneri [1]. It should be noted that, up to date, there is no complete protocol for the analysis and description of bacterial cell images obtained by the scanning probe microscopy.
The scanning probe microscopy provides a wide range of tools for studying bacterial cells. Its instruments include: obtaining three-dimensional images with nanoscale details, mapping of mechanical local surface properties – stiffness, friction, adhesion, wear resistance, fracture resistance. Observations with live bacteria can be performed dynamically both in air and in liquid.
In this paper we attempted to develop a software functionality for morphological analysis of bacterial cells. The developed functionality presents a module built-in in the FemtoScan Online software which performs the following basic quantitative measurements:
determination of the geometric dimensions of objects: length, width, height, area, volume;
calculation of contour length and roughness of objects;
determination of surface roughness parameters of objects;
determination of heterogeneity of microbial and cellular objects;
characterization of cell outgrowths in bacterial cells;
construction of table and histogram of distribution of measured morphological parameters of objects.
The functionality was tested mainly with bacterial Escherichia coli cells. Figure 2 shows a colony of E. coli bacteria and a histogram of the particle height distribution, Fig. 3 shows a measurement of the contour length of an E. coli outgrowth.
Additionally, the analysis was carried out on Klebsiella pneumoniae bacteria. Figure 4 shows a measurement of the surface roughness of Klebsiella pneumoniae bacteria. The surface roughness analysis is performed by measuring roughness on a baseline or a plane surface. The Ra parameter indicates the arithmetic mean deviation of the profile from the mean sloping line or plane, but this parameter is not sufficiently informative because surfaces can have the same mean roughness but have different shapes.
Therefore, the parameter Rq is often used, which indicates the standard deviation of the surface profile relative to the baseline. Rmax reflects the distance between the largest depression and the largest peak on the baseline. Rz measures the roughness of the profile at 10 points (5 largest troughs and 5 largest peaks). In the case of Klebsiella pneumoniae bacteria it is –0.005 nm. The asymmetry parameter Rsk shows the probability distribution that the profile has a given height Z. In our case the index is negative, which is characteristic of a surface with clear deep troughs on a smooth plateau.
The object search command allows you to find objects in the image and characterise them. Figure 5 shows an image of the found Klebsiella pneumoniae object. The values of perimeter, area, volume, variance of object height and form factor are automatically generated (Table 2).
CONCLUSIONS
The functionality presented in this paper provides a quantitative characteristic of the bacterial strain under study. At the same time, a visual emotional perception of the obtained images is an important component of the observations. In this connection, the software FemtoScan Online [2–5] opens up great opportunities, among them the construction of images of bacteria in different colour palettes for topography (top view) and 3D images with adjustable viewing angles, backlight modes and zooming. The software allows to perform editing of the fly over bacteria, thereby creating fascinating video clips. But that should be the subject of a separate story.
ACKNOWLEDGMENTS
The study was completed with the financial support of the RFBR and the London Royal Society No. 21-58-10005, and RFBR, Project No. 20-32-90036; this research was carried out with financial support from the Foundation for the Promotion of Innovation, Project No. 71108, and Agreement No. 0071108 and with the assistance of Endor LLC (Moscow, Russia).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
As an example, let us consider the cases that are quite rare, but which cannot theoretically be omitted. Let us assume that there were only non-viable or dead bacteria in the sample and in the body itself, which may no longer cause harm to the body. A PCR test detects DNA and signals a presence of dangerous pathogens. The doctor prescribes a course of antibiotics as recommended, which, in this case, is useless and can be harmful. Another possibility is that the bacteria are still alive, but they have lost some of their enzyme activity due to the treatment they have already received. Again, the genetic test signals a danger that no longer exists.
Whole DNA can be found in a mammoth that is 5 million years old. The DNA is there, but there is no living mammoth. Perhaps in future scientists will correct this gap. In this example it turns out that the mere presence of DNA in the sample taken does not yet indicate a presence of the carrier itself.
There is a legitimate question. Will all bacteria with the same genotype also have the same phenotype? In other words, are twin bacteria exact copies of each other? Probe microscopy provides an answer to this question. If you take a single bacterium and grow a colony on nutrient medium, it is easy to see that the bacteria in the colony are of different sizes and slightly different shapes (see Fig.1). It often happens that the larger bacteria grow on the edges of the colony. Strictly speaking, no two bacteria are exactly alike in a sea of bacteria. The reverse situation is often found in twins. It is a complete or very close external resemblance.
Which bacteria are potentially dangerous to humans? It is of the kind that scientists have already put on the list of dangerous bacteria. But for a bacterium to become really dangerous, it has to live a full life, including moving and multiplying by fission. For example, every 20–40 minutes. Such pathogenic bacteria pose the greatest danger to the development of disease.
Is it necessary to study the phenotype of bacteria, e.g. appearance, structure, features, etc.? What is more, not a static phenotype, but a phenotype in dynamics, in development. A complete description of a bacterium should include a description of the genotype (genetic protocol) and a description of the phenotype in living. It is the second description that the scanning probe microscopy allows.
RESEARCH METHODS
Since 1996, the probe microscopy has become an important tool for observation of bacterial cells. Even at that time in collaboration with the Gamaleya Institute (Gamaleya Research Center for EMS in the Russian Ministry of Health), it was possible to detect a difference in the parental strain of Escherichia coli and the transductant strain obtained from the parental strain by adding the rfb-a3,4 gene from Shigella flexneri [1]. It should be noted that, up to date, there is no complete protocol for the analysis and description of bacterial cell images obtained by the scanning probe microscopy.
The scanning probe microscopy provides a wide range of tools for studying bacterial cells. Its instruments include: obtaining three-dimensional images with nanoscale details, mapping of mechanical local surface properties – stiffness, friction, adhesion, wear resistance, fracture resistance. Observations with live bacteria can be performed dynamically both in air and in liquid.
In this paper we attempted to develop a software functionality for morphological analysis of bacterial cells. The developed functionality presents a module built-in in the FemtoScan Online software which performs the following basic quantitative measurements:
determination of the geometric dimensions of objects: length, width, height, area, volume;
calculation of contour length and roughness of objects;
determination of surface roughness parameters of objects;
determination of heterogeneity of microbial and cellular objects;
characterization of cell outgrowths in bacterial cells;
construction of table and histogram of distribution of measured morphological parameters of objects.
The functionality was tested mainly with bacterial Escherichia coli cells. Figure 2 shows a colony of E. coli bacteria and a histogram of the particle height distribution, Fig. 3 shows a measurement of the contour length of an E. coli outgrowth.
Additionally, the analysis was carried out on Klebsiella pneumoniae bacteria. Figure 4 shows a measurement of the surface roughness of Klebsiella pneumoniae bacteria. The surface roughness analysis is performed by measuring roughness on a baseline or a plane surface. The Ra parameter indicates the arithmetic mean deviation of the profile from the mean sloping line or plane, but this parameter is not sufficiently informative because surfaces can have the same mean roughness but have different shapes.
Therefore, the parameter Rq is often used, which indicates the standard deviation of the surface profile relative to the baseline. Rmax reflects the distance between the largest depression and the largest peak on the baseline. Rz measures the roughness of the profile at 10 points (5 largest troughs and 5 largest peaks). In the case of Klebsiella pneumoniae bacteria it is –0.005 nm. The asymmetry parameter Rsk shows the probability distribution that the profile has a given height Z. In our case the index is negative, which is characteristic of a surface with clear deep troughs on a smooth plateau.
The object search command allows you to find objects in the image and characterise them. Figure 5 shows an image of the found Klebsiella pneumoniae object. The values of perimeter, area, volume, variance of object height and form factor are automatically generated (Table 2).
CONCLUSIONS
The functionality presented in this paper provides a quantitative characteristic of the bacterial strain under study. At the same time, a visual emotional perception of the obtained images is an important component of the observations. In this connection, the software FemtoScan Online [2–5] opens up great opportunities, among them the construction of images of bacteria in different colour palettes for topography (top view) and 3D images with adjustable viewing angles, backlight modes and zooming. The software allows to perform editing of the fly over bacteria, thereby creating fascinating video clips. But that should be the subject of a separate story.
ACKNOWLEDGMENTS
The study was completed with the financial support of the RFBR and the London Royal Society No. 21-58-10005, and RFBR, Project No. 20-32-90036; this research was carried out with financial support from the Foundation for the Promotion of Innovation, Project No. 71108, and Agreement No. 0071108 and with the assistance of Endor LLC (Moscow, Russia).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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