Conceived and designed the experiments: EB FJ SV MR. Performed the experiments: EB FJ SV CJ M-FdlC JC MR. Analyzed the data: EB FJ SV MR. Contributed reagents/materials/analysis tools: M-FdlC. Wrote the paper: EB FJ SV CJ MR.
The authors have declared that no competing interests exist.
The diffusion of antibiotics in endocarditis vegetation bacterial masses has not been described, although it may influence the efficacy of antibiotic therapy in endocarditis. The objective of this work was to assess the diffusion of ofloxacin in experimental endocarditis vegetation bacterial masses using synchrotron-radiation UV fluorescence microspectroscopy. Streptococcal endocarditis was induced in 5 rabbits. Three animals received an unique IV injection of 150 mg/kg ofloxacin, and 2 control rabbits were left untreated. Two fluorescence microscopes were coupled to a synchrotron beam for excitation at 275 nm. A spectral microscope collected fluorescence spectra between 285 and 550 nm. A second, full field microscope was used with bandpass filters at 510–560 nm. Spectra of ofloxacin-treated vegetations presented higher fluorescence between 390 and 540 nm than control. Full field imaging showed that ofloxacin increased fluorescence between 510 and 560 nm. Ofloxacin diffused into vegetation bacterial masses, although it accumulated in their immediate neighborhood. Fluorescence images additionally suggested an ofloxacin concentration gradient between the vegetation peripheral and central areas. In conclusion, ofloxacin diffuses into vegetation bacterial masses, but it accumulates in their immediate neighborhood. Synchrotron radiation UV fluorescence microscopy is a new tool for assessment of antibiotic diffusion in the endocarditis vegetation bacterial masses.
Bacterial endocarditis is a severe infection developing at the surface of the cardiac
valvular apparatus. The endocarditis lesion, the so-ca lled vegetation, is mainly
composed of bacterial masses embedded in a platelet and plasma proteins matrix
Fluorescence microscopy has previously been used to study the diffusion of
tetracyclins in bacterial biofilm and uninfected bone thanks to their particular
fluorescence properties compatible with epifluorescence microscopy and confocal
scanning laser microscopy
The diffusion of ofloxacin in the endocarditis vegetation has not been reported.
Ofloxacin has interesting autofluorescence properties, with emission wavelengths
ranging from 400 to 600 nm after excitation at 275 nm
Ofloxacin in PBS (pH 7.4) showed a maximal fluorescence peak at 460 nm (range
390–600 nm) as shown in
Ofloxacin concentrations in PBS and serum were respectively 140 and 212 µmom/L (51 and 76 mg/L). Serum had a high intrinsic autofluorescence at 340 nm.
For rabbits treated by 150 mg/kg ofloxacin, ofloxacin mean ± SD concentrations were 170±16 µmol/L (61±6 mg/L) in serum and 241±27 nmol/g (87±10 µg/g) in vegetation.
Spectra extracted from vegetation maps of 3 ofloxacin-treated animals and 2 control rabbits were stratified on the 339 nm peak intensity by treatment status (control or ofloxacin), and by tissue class (bacterial mass or surrounding vegetation tissue). Then, selected spectra (control, n = 962; ofloxacin-treated, n = 962) were analyzed by Principal Component Analysis (PCA).
The first and second principal components (PC1 and PC2) accounted respectively
for 89.5% and 7.8% of the total spectral variance. The score plot
of PC1 and PC2 showed that the PC2 discriminated control and ofloxacin spectra
(
Score plot of first (PC1) and second (PC2) principal components (A). Control (blue +) and ofloxacin-treated (red o) spectra were respectively associated with positive and negative PC2 scores. Correlation loading plot of PC1 and PC2 (B). Correlation of wavelengths with PC1 and PC2 scores are shown for wavelengths ranging from 290 to 540 nm. The outer ellipse and inner ellipse indicate 100% and 50% explained variance respectively. C = control; O = ofloxacin.
After examination of mean control and ofloxacin spectra (
Bold and thin lines represent respectively mean and borders of the 95% confidence interval. Confidence interval border lines may superimpose on mean lines.
Spectral range (nm) | 390–440 a | 440–540 a | 510–560 b | |
|
|
774 (743–804)c | 2879 (2810–2946)c | 3824 (3745–3904)c |
|
855 (809–902)c | 3694 (3588–3799)c | 9828 (9782–9873)c | |
|
|
452 (431–473)c | 2151 (2114–2189)c | 5061 (5013–5109)c |
|
878 (819–939)c | 2955 (2869–3940)c | 9824 (9790–9859)c |
Mean (95% confidence interval) peak areas (arbitrary units)
were measured using spectral (a) and full-field (b) fluorescence
microscopes. Differences between control and ofloxacin (c) were all
significant (Mann-Whitney test,
In order to assess the diffusion of ofloxacin from surrounding vegetation into
bacterial masses, maps of the 390–440 nm peak area were obtained from
preprocessed spectra. Of note, spectra were not selected on the basis of their
339 nm band value as we did for PCA and previous statistics. Therefore,
fluorescence intensities of these maps were different from values reported in
The grayscale was the same for both fluorescence maps. White bar = 10 µm.
The full field microscope collected fluorescence in the 510–560 nm spectral
range (
Transmission (left) and fluorescence (right) images. The bacterial masses imaged on maps B, C and D located respectively in the intermediate, peripheral and central areas of the tissue specimen. The grayscale was the same for all maps. Fluorescence intensity values (median [range]) for A, B, C and D maps were respectively 2624 [0–14560], 8160 [1168–23376], 6736 [0–34928], and 6128 [432, 14640]. Each ofloxacin map intensity was significantly different from the control map (Mann-Whitney test, p<0.0001 for all). White bar = 10 µm.
Fluorescence of ofloxacin bacterial masses and surrounding vegetation were very
similar, indicating that ofloxacin diffused from the surrounding tissue into
bacterial masses (
Furthermore, the highest fluorescence intensities were observed for bacterial
masses that located close to the border of the vegetation (
The
The position of the bacterial mass border was superimposed on the
fluorescence map of the 510–560 nm range. For transmission image,
see
This study demonstrates that synchrotron-radiation UV fluorescence microspectroscopy
detects ofloxacin in the endocarditis vegetation despite the autofluorescence of
vegetation itself. The vegetation autofluorescence showed emission peaks at 339 and
410 nm, respectively due to tryptophan and NADH
Ofloxacin increased fluorescence intensities between 390 and 540 nm both in serum and in vegetation, and between 510 and 560 nm in vegetation. We do not claim that fluorescence allows to assess exactly the tissue or serum concentration of ofloxacin, but experiments on serum suggest that tissue fluorescence between 390 and 540 nm was roughly proportional to the ofloxacin concentration.
Two deep UV microscopes have been used in this study. The first microscope collected fluorescence spectra of vegetations, and allowed us to show that control and ofloxacin treated vegetations had different fluorescence patterns between 390 and 540 nm. Map acquisition with this microscope typically spends 1 h for a 80×80 µm2 map, and optical design yields a maximal sensitivity for wavelengths ranging from 300 and 450 nm. The second, full-field microscope gives no spectral information, but global fluorescence intensities for a given spectral range (510–560 nm in this study). Advantages of this second microscope are (i) high sensitivity (about 1000-fold the sensitivity of the spectral microscope), especially in the >500 nm range, and (ii) very short acquisition time (about 1 s for a 200×200 µm2 area). Hence the two fluorescence microscopes used in this study were complementary. In contrast with autoradiographic studies, synchrotron-radiation UV fluorescence microscopy allows the use of non-tagged drugs and assessments at multiple treatment time points. To be eligible to synchrotron-radiation UV fluorescence microspectroscopic studies, antibiotics should emit fluorescence light after excitation between 200 and 400 nm, with minimal interference with vegetation autofluorence emission spectra.
Our experiments were designed to demonstrate that synchrotron radiation UV
fluorescence microspectrocopy detects ofloxacin in vegetation bacterial masses after
a single injection. To maximize fluorescence detection, we administered a 150 mg/kg
dose of ofloxacin, higher than doses previously used for experimental study of
ofloxacin activity
Ofloxacin accumulates in the immediate neighborhood of vegetation bacterial masses, although this gradient may equilibrate in longer experiments. Furthermore, our work shows that ofloxacin diffuses into vegetation bacterial masses as soon as 30 minutes after the end of an unique injection. The diffusion of antibiotics in the endocarditis vegetation bacterial masses has not been previously reported, although poor diffusion is a potential risk factor for antibiotic therapy failure. The diffusion in vegetation bacterial masses may vary between antibacterial agents, thus influencing their antibacterial activity in endocarditis. Furthermore, diffusion of antibiotics in vegetation bacterial masses may be influenced by bacterial factors (e.g. biofilm or capsule production), and by antibacterial therapy modalities (e.g. injection number and duration). Using the example of ofloxacin, we showed here that synchrotron-radiation UV fluorescence microspectroscopy is a new tool to study the diffusion of antibacterial agents in infectious tissues and opens a new field of research in antibacterial chemotherapy.
Animal experiments were carried out in accordance with European Commission Directive 86/609/EEC, and were approved by the committee of animal ethics of the University of Nantes (approval C44015). All surgery was performed under ketamine and diazepam anesthesia, and all efforts were made to minimize suffering.
New Zealand white rabbits (weight: 2.0 to 2.7 kg) were kept in cages with free
access to food and water. A polyethylene catheter was inserted into the left
ventricle via the carotid artery and left in place throughout the experiment.
Three additional rabbits were treated with 150 mg/kg ofloxacin to assess
ofloxacin concentration in homogeneized vegetations. To assess the relationship
between serum fluorescence and ofloxacin concentration, 4 rabbits were treated
with lower doses of ofloxacin (20 and 120 mg/kg). Ofloxacin concentrations in
serum and in homogeneized vegetations were assessed by bioassay. Standard curves
for homogeneized vegetation and in sera were constructed respectively in PBS and
in serum, as previously described
Bacterial masses were localized under visible light in confrontation with the HE
stained contiguous slices. Hence, each pixel, and consequently each fluorescence
spectrum, could be classified in either bacterial mass or surrounding tissue.
Two UV microscopes directly coupled to the synchrotron beam at DISCO Beamline,
Synchrotron SOLEIL, were used. The first, spectral microscope was constructed
around an Olympus IX71 inverted microscope as previously described and used with
a Zeiss Ultrafluar 40x objective
The second, full field imaging system was a Zeiss Axio observer microscope with a
Zeiss Ultrafluar 40x objective. Dichroic mirrors with 50%
transmission/reflexion at 300 nm (Omega Optical, Brattleboro, Vermont) reflected
the incident light from DISCO bending magnet onto the sample
The spectral microscope was also used to study the fluorescence of ofloxacin in PBS and rabbit serum. A 140 µmol/L solution of ofloxacin in PBS (pH 7.4) was deposited in glass cupules closed by quartz coverslips and examined at λexc of 275 nm. The same experiment was done with control and ofloxacin-treated rabbit sera to determine if ofloxacin fluorescence in serum correlates with its concentration.
Spectra were spike filtered and noise was corrected by a Fourier-Transform filter using a home made Matlab procedure (Marie-Françoise Devaux, Institut National de la Recherche Agronomique, Nantes, France). To compare control and ofloxacin spectra acquired from different maps, an offset was applied to all spectra in order to set the count at 288 nm to zero. Spectra with less than 100 counts at 339 nm were excluded in order to optimize the signal to noise ratio. Wavelengths superior to 540 nm were excluded in order to exclude the Rayleigh band harmonics. We compared control and ofloxacin-treated spectra after stratification on the fluorescence intensity at 339 nm - the main peak of fluorescence emission spectra, because fluorescence at 339 nm slightly varied between maps, due to experimental factors as slice thickness, microscope focusing and alignment, and photobleaching.
All map processing, spectra preprocessing and analysis were performed with Matlab
R2007b and the SAISIR 2008 package of functions for chemometrics in the
Matlab* environment (