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Bacteria tracking by in vivo magnetic resonance imaging.

Hoerr V, Tuchscherr L, Hüve J, Nippe N, Loser K, Glyvuk N, Tsytsyura Y, Holtkamp M, Sunderkötter C, Karst U, Klingauf J, Peters G, Löffler B, Faber C - BMC Biol. (2013)

Bottom Line: The key step for successful labeling was to manipulate the bacterial surface charge by producing electro-competent cells enabling charge interactions between the iron particles and the cell wall.With 5-nm citrate-coated particles an iron load of 0.015 ± 0.002 pg Fe/bacterial cell was achieved for Staphylococcus aureus.The established cell labeling strategy can easily be transferred to other bacterial species and thus provides a conceptual advance in the field of molecular MRI.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Radiology, University Hospital Münster, Münster 48149, Germany.

ABSTRACT

Background: Different non-invasive real-time imaging techniques have been developed over the last decades to study bacterial pathogenic mechanisms in mouse models by following infections over a time course. In vivo investigations of bacterial infections previously relied mostly on bioluminescence imaging (BLI), which is able to localize metabolically active bacteria, but provides no data on the status of the involved organs in the infected host organism. In this study we established an in vivo imaging platform by magnetic resonance imaging (MRI) for tracking bacteria in mouse models of infection to study infection biology of clinically relevant bacteria.

Results: We have developed a method to label Gram-positive and Gram-negative bacteria with iron oxide nano particles and detected and pursued these with MRI. The key step for successful labeling was to manipulate the bacterial surface charge by producing electro-competent cells enabling charge interactions between the iron particles and the cell wall. Different particle sizes and coatings were tested for their ability to attach to the cell wall and possible labeling mechanisms were elaborated by comparing Gram-positive and -negative bacterial characteristics. With 5-nm citrate-coated particles an iron load of 0.015 ± 0.002 pg Fe/bacterial cell was achieved for Staphylococcus aureus. In both a subcutaneous and a systemic infection model induced by iron-labeled S. aureus bacteria, high resolution MR images allowed for bacterial tracking and provided information on the morphology of organs and the inflammatory response.

Conclusion: Labeled with iron oxide particles, in vivo detection of small S. aureus colonies in infection models is feasible by MRI and provides a versatile tool to follow bacterial infections in vivo. The established cell labeling strategy can easily be transferred to other bacterial species and thus provides a conceptual advance in the field of molecular MRI.

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Iron dilution during cell growth determined by MRI. (A) Cell density and (B) transverse relaxation time (T2) of iron-labeled S. aureus samples at different time points of the growth curve. Cell cultures were labeled with 5-nm citrate coated IONPs (1 μmol Fe/ml for 108 cells). For MRI measurements bacterial samples at each time point were diluted to an initial cell concentration of OD 0.5 and were embedded in 500 μl 0.5% agarose. (C) T2 measurements of agarose embedded cell cultures labeled with different iron concentrations in a 1:2 dilution row: 1 μmol Fe/ml, 0.5 μmol Fe/ml, 0.25 μmol Fe/ml, 0.125 μmol Fe/ml, 0.063 μmol Fe/ml, 0.031 μmol Fe/ml, 0.016 μmol Fe/ml. 1:2 dilution steps of the initial iron concentration imitate the iron dilution caused by cell division cycles. Note that dilution step 5 in (C) corresponds with measurement at 3 h in (B).
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Figure 3: Iron dilution during cell growth determined by MRI. (A) Cell density and (B) transverse relaxation time (T2) of iron-labeled S. aureus samples at different time points of the growth curve. Cell cultures were labeled with 5-nm citrate coated IONPs (1 μmol Fe/ml for 108 cells). For MRI measurements bacterial samples at each time point were diluted to an initial cell concentration of OD 0.5 and were embedded in 500 μl 0.5% agarose. (C) T2 measurements of agarose embedded cell cultures labeled with different iron concentrations in a 1:2 dilution row: 1 μmol Fe/ml, 0.5 μmol Fe/ml, 0.25 μmol Fe/ml, 0.125 μmol Fe/ml, 0.063 μmol Fe/ml, 0.031 μmol Fe/ml, 0.016 μmol Fe/ml. 1:2 dilution steps of the initial iron concentration imitate the iron dilution caused by cell division cycles. Note that dilution step 5 in (C) corresponds with measurement at 3 h in (B).

Mentions: For the use of MRI to detect and track bacteria in animal models, it is of the utmost importance that the label is conserved over time and bacteria remain detectable after several growth cycles. Electron microscopy showed that IONPs remained bound to the surface after cell division and were passed on to the daughter cells (Figure 1F). Thus, each cell division step continuously led to a dilution of IONPs. To determine how fast the iron on the cell wall is diluted during cell growth and to estimate the minimum iron concentration that is detectable by MRI, growth curves of labeled bacteria were measured using an initial inoculum of an OD (optical density) value of 0.5. Every hour OD values and CFU (colony forming units) counts were determined and corresponding T1 (longitudinal relaxation time) and T2 (transverse relaxation time) values were measured at 9.4 T from a sample diluted to the initial cell concentration of 0.5 OD. Prior to MRI measurements the corresponding cell samples were separated from the medium and were embedded in 500 μl of 0.5% agarose. While T1 did not change significantly over the time course, T2 was significantly decreased at the beginning of the growth curve and increased steadily until a stationary value was reached after 3 h (Figure 3A, B, Additional file 4). According to the OD values and CFU counts, the cell density increased 35-fold during the first 3 h which resulted in a cell concentration of 2.3 × 109 cells/ml. This observation confirmed that the binding of IONPs to the cell surface is stable and can be detected at least over five cycles of cell division. To assess the sensitivity and reliability of detection, we reversed the culture experiment and measured T2 of bacteria cultures labeled with a serial two-fold dilution of IONPs (Figure 3C). The corresponding T2 values indicated that bacteria were labeled to saturation for the initial dilution steps. From the third dilution on, T2 increased continuously with decreasing amount of iron. The T2 relaxation times measured after different cell division cycles (Figure 3B) correlated well with those obtained from cells labeled with different iron dilutions. T2 relaxation times of 121 ms and 142 ms, which were measured with a 1:8 and 1:32 iron dilution were also obtained from the growth curve at 1.2 h and 3 h, perfectly corresponding to three and five cell divisions.


Bacteria tracking by in vivo magnetic resonance imaging.

Hoerr V, Tuchscherr L, Hüve J, Nippe N, Loser K, Glyvuk N, Tsytsyura Y, Holtkamp M, Sunderkötter C, Karst U, Klingauf J, Peters G, Löffler B, Faber C - BMC Biol. (2013)

Iron dilution during cell growth determined by MRI. (A) Cell density and (B) transverse relaxation time (T2) of iron-labeled S. aureus samples at different time points of the growth curve. Cell cultures were labeled with 5-nm citrate coated IONPs (1 μmol Fe/ml for 108 cells). For MRI measurements bacterial samples at each time point were diluted to an initial cell concentration of OD 0.5 and were embedded in 500 μl 0.5% agarose. (C) T2 measurements of agarose embedded cell cultures labeled with different iron concentrations in a 1:2 dilution row: 1 μmol Fe/ml, 0.5 μmol Fe/ml, 0.25 μmol Fe/ml, 0.125 μmol Fe/ml, 0.063 μmol Fe/ml, 0.031 μmol Fe/ml, 0.016 μmol Fe/ml. 1:2 dilution steps of the initial iron concentration imitate the iron dilution caused by cell division cycles. Note that dilution step 5 in (C) corresponds with measurement at 3 h in (B).
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Related In: Results  -  Collection

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Figure 3: Iron dilution during cell growth determined by MRI. (A) Cell density and (B) transverse relaxation time (T2) of iron-labeled S. aureus samples at different time points of the growth curve. Cell cultures were labeled with 5-nm citrate coated IONPs (1 μmol Fe/ml for 108 cells). For MRI measurements bacterial samples at each time point were diluted to an initial cell concentration of OD 0.5 and were embedded in 500 μl 0.5% agarose. (C) T2 measurements of agarose embedded cell cultures labeled with different iron concentrations in a 1:2 dilution row: 1 μmol Fe/ml, 0.5 μmol Fe/ml, 0.25 μmol Fe/ml, 0.125 μmol Fe/ml, 0.063 μmol Fe/ml, 0.031 μmol Fe/ml, 0.016 μmol Fe/ml. 1:2 dilution steps of the initial iron concentration imitate the iron dilution caused by cell division cycles. Note that dilution step 5 in (C) corresponds with measurement at 3 h in (B).
Mentions: For the use of MRI to detect and track bacteria in animal models, it is of the utmost importance that the label is conserved over time and bacteria remain detectable after several growth cycles. Electron microscopy showed that IONPs remained bound to the surface after cell division and were passed on to the daughter cells (Figure 1F). Thus, each cell division step continuously led to a dilution of IONPs. To determine how fast the iron on the cell wall is diluted during cell growth and to estimate the minimum iron concentration that is detectable by MRI, growth curves of labeled bacteria were measured using an initial inoculum of an OD (optical density) value of 0.5. Every hour OD values and CFU (colony forming units) counts were determined and corresponding T1 (longitudinal relaxation time) and T2 (transverse relaxation time) values were measured at 9.4 T from a sample diluted to the initial cell concentration of 0.5 OD. Prior to MRI measurements the corresponding cell samples were separated from the medium and were embedded in 500 μl of 0.5% agarose. While T1 did not change significantly over the time course, T2 was significantly decreased at the beginning of the growth curve and increased steadily until a stationary value was reached after 3 h (Figure 3A, B, Additional file 4). According to the OD values and CFU counts, the cell density increased 35-fold during the first 3 h which resulted in a cell concentration of 2.3 × 109 cells/ml. This observation confirmed that the binding of IONPs to the cell surface is stable and can be detected at least over five cycles of cell division. To assess the sensitivity and reliability of detection, we reversed the culture experiment and measured T2 of bacteria cultures labeled with a serial two-fold dilution of IONPs (Figure 3C). The corresponding T2 values indicated that bacteria were labeled to saturation for the initial dilution steps. From the third dilution on, T2 increased continuously with decreasing amount of iron. The T2 relaxation times measured after different cell division cycles (Figure 3B) correlated well with those obtained from cells labeled with different iron dilutions. T2 relaxation times of 121 ms and 142 ms, which were measured with a 1:8 and 1:32 iron dilution were also obtained from the growth curve at 1.2 h and 3 h, perfectly corresponding to three and five cell divisions.

Bottom Line: The key step for successful labeling was to manipulate the bacterial surface charge by producing electro-competent cells enabling charge interactions between the iron particles and the cell wall.With 5-nm citrate-coated particles an iron load of 0.015 ± 0.002 pg Fe/bacterial cell was achieved for Staphylococcus aureus.The established cell labeling strategy can easily be transferred to other bacterial species and thus provides a conceptual advance in the field of molecular MRI.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Radiology, University Hospital Münster, Münster 48149, Germany.

ABSTRACT

Background: Different non-invasive real-time imaging techniques have been developed over the last decades to study bacterial pathogenic mechanisms in mouse models by following infections over a time course. In vivo investigations of bacterial infections previously relied mostly on bioluminescence imaging (BLI), which is able to localize metabolically active bacteria, but provides no data on the status of the involved organs in the infected host organism. In this study we established an in vivo imaging platform by magnetic resonance imaging (MRI) for tracking bacteria in mouse models of infection to study infection biology of clinically relevant bacteria.

Results: We have developed a method to label Gram-positive and Gram-negative bacteria with iron oxide nano particles and detected and pursued these with MRI. The key step for successful labeling was to manipulate the bacterial surface charge by producing electro-competent cells enabling charge interactions between the iron particles and the cell wall. Different particle sizes and coatings were tested for their ability to attach to the cell wall and possible labeling mechanisms were elaborated by comparing Gram-positive and -negative bacterial characteristics. With 5-nm citrate-coated particles an iron load of 0.015 ± 0.002 pg Fe/bacterial cell was achieved for Staphylococcus aureus. In both a subcutaneous and a systemic infection model induced by iron-labeled S. aureus bacteria, high resolution MR images allowed for bacterial tracking and provided information on the morphology of organs and the inflammatory response.

Conclusion: Labeled with iron oxide particles, in vivo detection of small S. aureus colonies in infection models is feasible by MRI and provides a versatile tool to follow bacterial infections in vivo. The established cell labeling strategy can easily be transferred to other bacterial species and thus provides a conceptual advance in the field of molecular MRI.

Show MeSH
Related in: MedlinePlus