Doppler imaging detects bacterial infection of living tissue

Abstract

Living 3D in vitro tissue cultures, grown from immortalized cell lines, act as living sentinels as pathogenic bacteria invade the tissue. The infection is reported through changes in the intracellular dynamics of the sentinel cells caused by the disruption of normal cellular function by the infecting bacteria. Here, the Doppler imaging of infected sentinels shows the dynamic characteristics of infections. Invasive Salmonella enterica serovar Enteritidis and Listeria monocytogenes penetrate through multicellular tumor spheroids, while non-invasive strains of Escherichia coli and Listeria innocua remain isolated outside the cells, generating different Doppler signatures. Phase distributions caused by intracellular transport display Lévy statistics, introducing a Lévy-alpha spectroscopy of bacterial invasion. Antibiotic treatment of infected spheroids, monitored through time-dependent Doppler shifts, can distinguish drug-resistant relative to non-resistant strains. This use of intracellular Doppler spectroscopy of living tissue sentinels opens a new class of microbial assay with potential importance for studying the emergence of antibiotic resistance.

Fig. 1. Biodynamic imaging system configuration.

a Experimental principles and setup of intracellular Doppler spectroscopic imaging. A living sample (tumor spheroid or biopsy) is optically sectioned with low-coherence backscattered infrared light on a digital camera on the Fourier plane using a Mach–Zehnder off-axis digital holographic system. The low-coherence light is split by a beam splitter (BS1) into the object and reference arms. Backscattered light is collected by the 4-f system (L1 and L2) and forms an image on the image plane (IP). The Fourier lens (L3) transfers the image onto the Fourier plane (FP). b Scattering of light from dynamic intracellular processes generates dynamic speckle that is altered when tumor spheroids are inoculated with various bacterial strains. c The Fourier-domain digital hologram is reconstructed to the image plane using a fast Fourier transform. The zero-order is in the center, and the two first-order side-bands are on the diagonal. The red box shows a magnified part of the image plane speckle. d A magnified reconstructed speckle image. e Dynamic speckle intensity fluctuates temporally at different locations P1, P2, and P3.

Fig. 2. Examples of DLD-1 multicellular tumor spheroids before and after infection.

a Optical coherence images (OCI) of the optical mid-sections from DLD-1 spheroids showing backscatter brightness before (left) and 6 h after (right) infection by 10⁷ bacterial CFU per well. The strongest increase in backscatter brightness (BB) occurs for E. coli (note second OCI column). b Motility contrast images (MCI) for the same samples. The growth of the bacteria causes a strong inhibition of Doppler activity for E. coli (note second MCI column) and moderate inhibition of Doppler activity for Listeria monocytogenes and Salmonella enterica serovar Enteritidis. Slowly proliferating Listeria innocua shows the smallest effect. The scale bar applies to all speckle images. c Temporal evolution in BB as a function of time after inoculation (red dashed line). Infection by E. coli produces a large increase in BB. d Dynamic range (DR) of the spectral density changes non-monotonically as a function of time. The error bars represent standard error.

Fig. 3. Spectral responses by various bacteria.

a Changes in the Doppler spectra (intensity normalized) for the four bacterial strains with initial loads of 10⁶ CFU per well. Mid frequencies are inhibited in all cases with the strongest effect from E. coli. The high-frequency Nyquist floor (related to organelle transport) increases noticeably for S. enterica. The values for N are the number of replicate samples used in the average. The uncertainty in spectral amplitudes is ±4%. b The associated spectrograms are generated using Eq. (2). The samples are inoculated (red dashed line) after a baseline is established. The contours (10 in each graph) help show the general trends.

Fig. 4. Representative cases of tissue-dynamics spectroscopic images (TDSI) of bacterial invasion of 10⁷ CFU into a DLD colon adenocarcinoma tumor spheroid.

a The linear filter used here measures red or blue shifts of spectral shape (zero-sum normalization). The untreated control displays a small blue shift (blue colored) in the outer layers (R1) and a small redshift (red-colored) in the core (R2) over the duration of the experiment (5 h). b L. innocua is indistinguishable from the control. S. enterica and L. monocytogenes display strong blue shifts throughout the volume within 300 m after infection. The blue shift for E. coli is intermediate between the pathogenic strains and the control. The scale bar applies to all other speckle images in b. c One-dimensional plots of TDSI along the axis L in a. The pathogen group (L. monocytogenes and S. enterica) display blue shifts throughout the volume. The y axis represents the relative change in average Doppler frequency (knee frequency of the spectrum) The two pathogenic strains induce approximately a 15% increase in intracellular speeds.

Fig. 5. Lévy statistics in living tissue and anomalous random walks associated with Levy flights.

a Lévy probabilities. Lévy processes with smaller alpha have more frequent ballistic motions and are less ballistic with larger alpha. b–d Examples of Lévy random walks displaying the heavy tail of persistence lengths. e The PDF of DLD-1 tissue with a Lévy exponent of α = 1.6. f The PDF of tissue infected by L. monocytogenes (10⁷ CFU/well), showing a measurable shift to α = 1.4 in only 1.5 h. g Temporal shifts of Levy exponents from multiple samples infected with the four strains of bacteria. Error bars represent standard error. A logistic function was used to fit the data.

Fig. 6. Time evolution of the spectral density of DLD-1 sentinels.

The top row shows the (intensity normalized) spectrogram responses for the infected DLD-1 sentinels. The dashed red line is the time of bacterial inoculation, and the dashed black line is the time of antibiotic application. The bottom row shows the responses of the DLD-1 sentinel controls without bacterial infection. E. coli infection induces broadband suppression of activity (top of left column). Treatment with ampicillin (middle column) has little effect on the infected tissue, but ciprofloxacin (right column) halts and reverses the infection, returning the tissue to a response similar to the uninfected control. The replicate numbers are shown at the top-left corner of the spectrogram plots.