Dorothea R Go ¨tte

Sylvie L Benestad

Hubert Laude

Andreas Zurbriggen

Anna Oevermann

Torsten Seuberlich
email: torsten.seuberlich@vetsuisse.unibe.ch

Atypical Scrapie Isolates Involve a Uniform Prion Species with a Complex Molecular Signature

The pathobiology of atypical scrapie, a prion disease affecting sheep and goats, is still poorly understood. In a previous study, we demonstrated that atypical scrapie affecting small ruminants in Switzerland differs in the neuroanatomical distribution of the pathological prion protein (PrP d ). To investigate whether these differences depend on host-related vs. pathogen-related factors, we transmitted atypical scrapie to transgenic mice over-expressing the ovine prion protein (tg338). The clinical, neuropathological, and molecular phenotype of tg338 mice is similar between mice carrying the Swiss atypical scrapie isolates and the Nor98, an atypical scrapie isolate from Norway. Together with published data, our results suggest that atypical scrapie is caused by a uniform type of prion, and that the observed phenotypic differences in small ruminants are likely host-dependant. Strikingly, by using a refined SDS-PAGE technique, we established that the prominent proteinase K-resistant prion protein fragment in atypical scrapie consists of two separate, unglycosylated peptides with molecular masses of roughly 5 and 8 kDa. These findings show similarities to those for other prion diseases in animals and humans, and lay the groundwork for future comparative research.

Introduction

Transmissible spongiform encephalopathies (TSEs), such as scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans, are fatal neurodegenerative diseases that are caused by prions, which are infectious misfolded proteins [START_REF] Prusiner | Molecular biology of prion diseases[END_REF]. The neuropathology of the TSEs includes spongiform vacuolation, gliosis, and the aggregation of a pathological isoform, PrP d , of the endogenous host prion protein, PrP c , in the brain. According to the proteinonly hypothesis, the PrP d isoform is the infectious agent [START_REF] Griffith | Self-replication and scrapie[END_REF]. PrP d differs biochemically from PrP c in a number of its characteristics, which include the partial resistance to proteolytic degradation by proteinase K (PK). PK-resistant PrP d fragments (PrP res ) can be detected via immunochemical techniques such as Western blotting (WB) [START_REF] Hope | The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP)[END_REF].

To date, three types of TSE have been found in small ruminants: classical scrapie, BSE, and atypical scrapie. Classical scrapie has been observed for more than two centuries [START_REF] Schneider | The early history of the transmissible spongiform encephalopathies exemplified by scrapie[END_REF]. Classical scrapie prions are transmitted between animals and via contamination of the environment, and may cause significant losses in affected small ruminant flocks. Experimentally, sheep and goats are susceptible to oral infection with BSE prions [START_REF] Foster | Transmission of bovine spongiform encephalopathy to sheep and goats[END_REF] and, recently, two goats have been described that were likely to have been naturally infected in the course of a BSE epidemic in cattle in Europe [START_REF] Eloit | BSE agent signatures in a goat[END_REF][START_REF] Jeffrey | Immunohistochemical features of PrP(d) accumulation in natural and experimental goat transmissible spongiform encephalopathies[END_REF]. For this reason, TSEs in small ruminants have been intensively monitored in European Union member states, however, to date, no further small ruminant BSE cases have been identified. Atypical scrapie was first observed in Norway in 1998 (hence, Nor98) and was later detected in a number of other countries primarily by means of active disease surveillance schemes [START_REF] Benestad | Cases of scrapie with unusual features in Norway and designation of a new type, Nor98[END_REF][START_REF] Benestad | Atypical/Nor98 scrapie: properties of the agent, genetics, and epidemiology[END_REF]. Such cases revealed discordant phenotypic features, in particular, SDS-PAGE PrP res banding patterns and neuroanatomical PrP d distributions that differ from those of classical scrapie and BSE. Moreover, these distinctions were frequently found in sheep that displayed prion protein genotypes associated with a relative resistance to classical scrapie, and there was rarely more than one animal per herd affected [START_REF] Fediaevsky | Discrepant epidemiological patterns between classical and atypical scrapie in sheep flocks under French TSE control measures[END_REF][START_REF] Luhken | Epidemiological and genetical differences between classical and atypical scrapie cases[END_REF].

Upon transmission into sheep and rodents models in the laboratory, this distinct phenotype was preserved, and it was concluded that atypical scrapie cases are caused by prion species different from those that underlie classical scrapie and BSE [START_REF] Arsac | Transmissibility of atypical scrapie in ovine transgenic mice: major effects of host prion protein expression and donor prion genotype[END_REF][START_REF] Dur | A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes[END_REF][START_REF] Simmons | Experimental transmission of atypical scrapie to sheep[END_REF][START_REF] Simmons | The natural atypical scrapie phenotype is preserved on experimental transmission and sub-passage in PRNP homologous sheep[END_REF][START_REF] Simmons | Experimental oral transmission of atypical scrapie to sheep[END_REF]. However, the pathobiology and phenotypic diversity of naturally occurring atypical scrapie remain to be elucidated. This lack of clarity results partly from the limited brain tissue available to researchers via active disease surveillance programs.

In a previous study, we had access to whole brains from small ruminants affected with atypical scrapie, and discovered a marked diversity in the neuroanatomical distribution of the PrP d ( [START_REF] Nentwig | Diversity in Neuroanatomical Distribution of Abnormal Prion Protein in Atypical Scrapie[END_REF], table 1). Similar findings were later reported by other groups in both naturally-occurring cases and following experimental oral transmission of atypical scrapie isolates to sheep [START_REF] Simmons | Experimental oral transmission of atypical scrapie to sheep[END_REF][START_REF] Wemheuer | Detection of classical and atypical/Nor98 scrapie by the paraffin-embedded tissue blot method[END_REF][START_REF] Rodriguez-Martinez | Atypical/Nor98 scrapie in the Basque Country: a case report of eight outbreaks[END_REF][START_REF] Wemheuer | PrPSc spreading patterns in the brain of sheep linked to different prion types[END_REF]. However, it remains to be determined whether this diversity in PrP d distribution is attributable to host factors, the involvement of particular prion types or a combination of both.

In the current report, we have addressed this question by transmitting a panel of eight small ruminant atypical scrapie isolates, with different neuroanatomical PrP d distribution patterns, into mice overexpressing the ovine prion protein. This mouse model has been shown to be highly susceptible to both classical and atypical scrapie prion pathology. We compared survival times, pathology attack rates, patterns of lesions, PrP d distribution in the brain, and biochemical PrP res characteristics between these different groups and with mice infected with isolates of classical scrapie and atypical/Nor98 scrapie. Our findings support that atypical scrapie, despite the phenotypic variations evident in the natural hosts, is caused by a uniform species of prion. Our characterization of atypical scrapie isolates also reveals a previously unrecognized complexity of the molecular PrP res phenotype.

Results

Clinical signs of disease, attack rates and survival times

Following inoculation with atypical/Nor98 and classical scrapie, mice consistently showed characteristic clinical signs of TSE. At the terminal stage of the disease, the animals displayed ataxia and tremor, lost weight, and exhibited impaired grooming behavior and kyphosis. There were no obvious differences in clinical presentation between the isolates studied. As some of the shaminoculated mice also showed neurological impairment at the end of their life-span, we defined mice as TSE positive or negative according to the presence or absence of PrP d in the brain in either immunohistochemistry (IHC) or WB. All eight Swiss atypical scrapie isolates, two derived from goats and six from sheep, and the Nor98 isolate transmitted efficiently to tg338 mice following intracerebral inoculation, with attack rates of 100% (Table 1). The average survival times in the atypical scrapie groups including the Nor98 were very similar (between 188 and 235 days) and did not differ significantly between groups that were infected with isolates from donor animals of different PRNP genotypes. However, inoculation survival in all atypical scrapie isolates and Nor98 was significantly shorter (p,0.0001) compared to the classical scrapie isolate (304617 days) and to the sham-inoculated mice (567622 days (Fig. 1)). as reported in our previous study [START_REF] Nentwig | Diversity in Neuroanatomical Distribution of Abnormal Prion Protein in Atypical Scrapie[END_REF]; -, structure missing; 1, mild; 2, moderate; 3, severe. *for S4/RS fourteen out of sixteen inoculated mice died within two days post inoculation, most probably due to a high bacterial contamination of the sample.

Histopathological brain lesion profiles

The intensity and neuroanatomical distribution of vacuolar lesions are very similar between mice infected with sheep (Fig. 2A) vs. goat (Fig. 2B) atypical scrapie isolates, and the Nor98 isolate. However, in the corpus callosum, a slight difference in the intensity of lesions is observed between mice infected with the Swiss atypical sheep isolates (Fig. 3B) and those with the Nor98 isolate (Fig. 2A). In addition, inoculation with the goat isolate G1/ RS results in lesion scores similar to the Nor98 isolate (Fig. 2B), while the inoculation with the sheep isolate S4/RS results in intermediate lesion scores (Fig. 2A) in the corpus callosum. Vacuolar lesions were, in general, less severe following inoculation with the classical scrapie isolate, although the hypothalamus still exhibited considerable lesions in these mice (Fig. 2C, Fig. 3A). In both, sham-and non-inoculated mice, we observed mild vacuolar lesions in most of the brain structures investigated, and lesions were particularly notable in the white matter of the cerebellum (Fig. 2D, Fig. 3C).

Neuroanatomical PrP d distribution

We assessed the distribution of the pathological PrP d protein at four rostro-caudal positions in the brains of inoculated mice (Fig. S1A). The distribution pattern of PrP d immunoreactivity is similar amongst the mice inoculated with the Swiss atypical scrapie isolates as well as the Nor98 isolate. However, the PrP d distribution in these groups is strikingly distinct from that observed in the classical scrapie-inoculated mice (Figs. 4,S1B). The localization and size of plaque-like PrP d deposits also differ greatly between the groups exposed to atypical vs. classical scrapie isolates (Fig. 5). These plaques are readily observable in hematoxylin and eosin-stained sections from mice inoculated to classical, but not atypical scrapie (Fig. 3A). In sham-inoculated mice, we observed a faint intraneuronal labeling in the cortex, the septal nuclei, and in some white matter structures, as well as a faint axonal staining in the thalamic nuclei (Figs. 4, S1B), the latter was also evident in non-inoculated tg338 mice (data not shown).

Biochemical characteristics of PrP res following transmission to tg338 mice

We used Western blotting to characterize the SDS-PAGE banding patterns of PrP res using our standard monoclonal antibody P4-based protocol [START_REF] Nentwig | Diversity in Neuroanatomical Distribution of Abnormal Prion Protein in Atypical Scrapie[END_REF] and 16.5% polyacrylamide gels. As expected, the PrP res WB patterns in the brains of the inoculated mice were similar with those previously published for classical and atypical scrapie, and consistent with the respective inocula. However, we identified an additional PrP res fragment with a molecular mass ,6.5 kDa in some immunoblots from mice inoculated with the atypical/Nor98 scrapie isolates (Fig. 6A). We then re-analyzed all the samples using 4-20% gradient tris-glycine polyacrylamide gels to better separate the low molecular mass proteins. These immunoblots were also probed with the P4 antibody and we found that all of the atypical scrapie samples, including the Nor98, showed the same pattern, with two distinct bands of lower molecular mass; one migrating at roughly 5 kDa, and another band at 8 kDa (Figs. 6B andS2). No bands were detected in the Western blot, when the P4 antibody was replaced by IgG purified from normal mouse serum (data not shown).

Sham-inoculated and non-inoculated control mice also showed some discrete PK-resistant bands in the Western blot. The bands were, in general, much less intense, visible only upon loading of relative large amounts of material and were not detected following more stringent PK-digestion (Fig. S3). However, in the majority of the sham-inoculated and non-inoculated mice we found an 8 kDa band of a relative high PK-resistance, which was reminiscent to that in the atypical scrapie infected mice (Figs. 6 B-E andS3).

Immunoblots containing representative atypical scrapie samples were then probed with the monoclonal Sha31 antibody, which is widely used in small ruminant TSE research and diagnostics and binds nearer to the C-terminus of the prion peptide than does P4 or the 9A2 antibody. After immunoblotting with Sha31, however, only the 8 kDa, but not the 5 kDa moiety of PrP res could be detected (Fig. 6C). We then treated the protein lysates with the endoglycosidase PNGase F, which removes N-linked glycosylation. Treatment with PNGase F indicated that the fragments migrating at 5 kDa, 8 kDa, and 18 kDa correspond to unglycosylated PrP res moieties, whereas those above 18 kDa are N-linked glycosylated. We found that immunoblotting with the 9A2 antibody resulted in the same pattern as P4, confirming the identity of these bands as PrP res fragments (Fig. 6D andE). While the ,18 kDa PrP res fragment and its glycoforms were relatively sensitive to more stringent PK-digestion, this was not the case for the 5 and 8 kDa peptides (Fig. 6F).

Biochemical PrP res characterization of sheep/goat isolates

We then investigated whether the low molecular mass dualbanding-pattern observed in mice inoculated with atypical scrapie was relevant to atypical scrapie in small ruminants. To this end, we used 4-20% gradient SDS-PAGE to analyze protein lysates obtained from the sheep and goat tissue used to prepare the atypical scrapie inocula. Interestingly, we found that the banding patterns obtained from immunoblotting with P4 in the atypical sheep and goat scrapie samples greatly resemble those obtained from atypical scrapie-inoculated tg338 mice. Similar to the findings with the inoculated tg338 mice, the 8 kDa peptide, but not the 5 kDa peptide, was detected using Sha31 (Fig. 7). Noteworthy, neither the 8 kDa peptide nor other PK-resistant bands were observed in TSE negative sheep (Figs. 7 andS3B). These results suggest that, in the natural host, both the 5 kDa and 8 kDa peptides are disease-specific, and that these fragments are C-terminally truncated to a different extent.

Discussion

The current study seeks to delineate the host-related from the pathogen-related factors that affect the phenotype of atypical scrapie in small ruminants. We show that the transmission of a panel of defined atypical scrapie isolates from sheep and goats in Switzerland, which displayed distinct neuroanatomical PrP d deposition patterns in the donor animals, resulted in a uniform disease phenotype in tg338 mice with respect to attack rates, survival times, PrP d distribution, and the molecular pattern of the PrP res peptides. Taken together, the overall phenotype of the Swiss atypical scrapie isolates was indistinguishable from that of the Nor98, but was clearly distinct from that of the classical scrapie isolate. Our data therefore corroborate previous findings that atypical scrapie involves a prion species distinct from those of classical scrapie. Moreover, our results indicate that the type of prion is identical in both the Nor98 isolate and the atypical scrapie cases presented in this work.

In our analysis of the severity and distribution of brain lesions, we observed some subtle differences between the various isolates. However, these differences fell within the range of the variability observed between individual mice within each isolate group, and are therefore not considered significant. An unexpected observation was that non-inoculated mice as well as sham-inoculated mice also displayed mild histopathological lesions and PrP immunore-activity. In the latter, this was complemented by neurological impairment at advanced age (i.e., 570 days post-inoculation) in some animals. These findings could be due to aging, sanitary status of the mouse colony or the housing conditions. Also, we cannot completely rule out that the negative inoculum contained lowlevels of prion infectivity. While this situation may hamper the interpretation of data when investigating prion isolates that reveal longer incubation periods, it does not impact on our ability to interpret the range of data produced with the atypical and classical scrapie isolates in the present study.

The phenotype of the Swiss atypical scrapie isolates in the tg338 model is strikingly similar to previously published data on atypical/Nor98 scrapie field isolates from Germany, Norway, France, and the UK that used both the tg338 mouse model as well Figure 2. Lesion profiles in scrapie-infected tg338 mice. Lesions were scored according to the protocol established by Fraser and Dickinson [START_REF] Fraser | The sequential development of the brain lesion of scrapie in three strains of mice[END_REF] in ten grey matter areas (left y-axis, scale 0 to 5) and in four white matter areas (right y-axis, scale 0 to 3). All depicted values represent means of scores in mice inoculated with the same isolate: atypical sheep scrapie (A), atypical goat scrapie (B), classical scrapie (C) as well as sham-inculated and non-inoculated mice (D). To facilitate cross-comparison, Nor98 scores are included in each graph. doi:10.1371/journal.pone.0027510.g002 as other ovine PrP transgenic mouse lines [START_REF] Groschup | Classic scrapie in sheep with the ARR/ARR prion genotype in Germany and France[END_REF][START_REF] Griffiths | Characterization of atypical scrapie cases from Great Britain in transgenic ovine PrP mice[END_REF][START_REF] Andreoletti | Atypical/Nor98 scrapie infectivity in sheep peripheral tissues[END_REF]. This suggests that there are no major differences between atypical scrapie prions isolated from various geographical origins and from sheep or goats. Arsac and collaborators [START_REF] Arsac | Transmissibility of atypical scrapie in ovine transgenic mice: major effects of host prion protein expression and donor prion genotype[END_REF] reported that atypical scrapie isolates from donor animals carrying PRNP alleles that were associated with a relative higher susceptibility to the disease (AF 141 RQ or AHQ) revealed a significantly shorter survival time compared to those derived from donor animals without such alleles in mice transgenic for the ovine ARQ PRNP allele. In our and others studies [START_REF] Dur | A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes[END_REF][START_REF] Griffiths | Characterization of atypical scrapie cases from Great Britain in transgenic ovine PrP mice[END_REF] a similar effect for atypical scrapie isolates was not observed in tg338 mice, which may be related to higher PrP expression levels and/or more rapid evolution of the disease in this model. Our results thus provide indirect evidence that the observed variability in the neuroanatomical PrP d distribution of atypical/Nor98 scrapie amongst small ruminants may result from, as yet unidentified, host factors rather than from the involvement of distinct prion agents. This situation contrasts sharply to that in classical scrapie, for which a large body of evidence points towards the existence of diverse prion strains, which differ in their phenotypic presentation in sheep and after transmission to mouse models [START_REF] Bruce | Strain Typing Studies of Scrapie and BSE[END_REF][START_REF] Dickinson | Scrapie pathogensis in inbred mice: an assessment of host control and response involving many strains of agent[END_REF][START_REF] Gonzalez | Variability in disease phenotypes within a single PRNP genotype suggests the existence of multiple natural sheep scrapie strains within Europe[END_REF]. It is conceivable that this strain diversity, as in other infectious diseases, results from serial transmission events and interactions with the host. In this respect, the classical scrapie isolate used in our study may not fully represent this class of TSE agents. However, the uniformity of the atypical scrapie isolates in the tg338 model, further questions the origin of this disease. Therefore, more data from transmission studies in small ruminants will be required to thoroughly characterize the etiology, pathogenesis and host-pathogen interaction in atypical scrapie. The molecular PrP res phenotypes in small ruminants and mice affected with atypical/Nor98 scrapie have been addressed in detail in a series of studies [START_REF] Arsac | Transmissibility of atypical scrapie in ovine transgenic mice: major effects of host prion protein expression and donor prion genotype[END_REF][START_REF] Griffiths | Characterization of atypical scrapie cases from Great Britain in transgenic ovine PrP mice[END_REF][START_REF] Klingeborn | Characterization of proteinase K-resistant N-and C-terminally truncated PrP in Nor98 atypical scrapie[END_REF][START_REF] Gretzschel | Immunological characterization of abnormal prion protein from atypical scrapie cases in sheep using a panel of monoclonal antibodies[END_REF][START_REF] Arsac | Similar biochemical signatures and prion protein genotypes in atypical scrapie and Nor98 cases, France and Norway[END_REF][START_REF] Everest | Atypical prion protein in sheep brain collected during the British scrapie-surveillance programme[END_REF]. In these studies there is some disagreement regarding the estimated molecular mass of the prominent PrP res band of lower molecular mass (,7-12 kDa), which is most likely due to different electrophoretic conditions. However, this band is universally considered to represent a single truncated PrP res fragment. Using gradient SDS-PAGE and a set of PrP-specific monoclonal antibodies, we now show that this band in fact involves two distinct PrP res fragments of approximately 8 kDa and 5 kDa. This dual-banding-pattern has likely been overlooked in the past, since to our knowledge all studies that focused on the molecular PrP res properties in atypical scrapie used 12-15% SDS PAGE, which do not resolve the 5 and 8 kDa peptides. Moreover, confirmatory Western blot techniques may miss the 5 kDa fragment, when relying on antibodies that bind to the center of the PrP res (,aminoacid 150), such as Sha31.

We detected the 8 kDa PrP res peptide in atypical/Nor98 scrapie-affected tg338 mice, sheep, and goats using the P4, 9A2, and Sha31 antibodies, indicating that this fragment includes at least amino acids 93 to 155 of the full-length mature prion protein. This band was detectable with a much lower signal in negative control mice, and exhibited a high resistance to proteolysis. However, the 8 kDa PrP res band was absent in TSE-negative sheep and clearly related to the atypical scrapie affected sheep and goats. This could be due to the over-expression of the ovine prion protein transgene in these mice, and may explain the relatively high susceptibility of these mice to atypical scrapie compared to other TSEs. In contrast, the 5 kDa PrP res species was observed only in atypical scrapie-affected tg338 mice and small ruminants, but not in the negative control mice and healthy small ruminants. The 5 kDa peptide also did not react with the more C-terminaldirected Sha31 antibody, and thus differs from the 8 kDa PrP res at its C-terminus.

Recently, we reported on a classical scrapie outbreak in a Greek goat flock, in which several animals exhibited a distinct C-and Nterminally ragged PK-resistant PrP res fragment [START_REF] Bouzalas | Distinct proteinase k-resistant prion protein fragment in goats with no signs of disease in a classical scrapie outbreak[END_REF]. This peptide demonstrated the same antibody-binding properties, and was estimated to be at similar molecular mass as the 5 kDa PrP res moiety described in the present study. At that time, it was unclear as to which type of prion disease this peptide was related. Our current findings support the idea that this fragment is related to atypical vs. classical scrapie, and that both prion species were present in the Greek flock. Future studies will conduct a direct comparison of these peptides by gradient SDS-PAGE, pending the receipt of material from mouse transmission studies of the Greek isolates.

PrP res peptides of low molecular mass have also been described in other types of prion disease, such as Gerstmann-Stra ¨ussler-Scheinker disease [START_REF] Chen | Truncated forms of the human prion protein in normal brain and in prion diseases[END_REF][START_REF] Tagliavini | A 7-kDa prion protein (PrP) fragment, an integral component of the PrP region required for infectivity, is the major amyloid protein in Gerstmann-Straussler-Scheinker disease A117V[END_REF] and Creutzfeldt-Jakob disease [START_REF] Zou | Identification of novel proteinase K-resistant C-terminal fragments of PrP in Creutzfeldt-Jakob disease[END_REF][START_REF] Krebs | A novel subtype of Creutzfeldt-Jakob disease characterized by a small 6 kDa PrP fragment[END_REF] in humans as well as in H-BSE in cattle [START_REF] Jacobs | Molecular discrimination of atypical bovine spongiform encephalopathy strains from a geographical region spanning a wide area in Europe[END_REF][START_REF] Langeveld | Four independent molecular prion protein parameters for discriminating new cases of C, L, and H BSE in cattle[END_REF]. Forthcoming directions of research are likely to focus on more precise comparative analyses of truncated PrP res peptides and their role in the biology of human and animal prion diseases.

Materials and Methods

TSE isolates

A total of eight atypical scrapie isolates from Switzerland [START_REF] Dur | A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes[END_REF][START_REF] Nentwig | Diversity in Neuroanatomical Distribution of Abnormal Prion Protein in Atypical Scrapie[END_REF] were included in this study, all of which had been identified by active or passive surveillance for TSEs in small ruminants in the years 2004 and 2005. An atypical scrapie isolate from Norway (Nor98), a classical scrapie isolate from an experimentally-infected sheep (genotype donor sheep VRQ/ VRQ; recipient VRQ/ARQ). A pool of medulla oblongata tissues from sheep that tested negative for TSEs by two independent TSE screening tests, and by immunohistochemistry (IHC) in different brain structures and lymphatic tissues, served as a sham-inoculum. Details on the isolates and controls are shown in Table 1.

Transgenic mice and bioassays

Tg338 mice express the ovine VRQ PrP on a murine PrP knock-out background at 6-to 8-fold higher levels compared to sheep [START_REF] Dur | A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes[END_REF][START_REF] Laude | New in vivo and ex vivo models for the experimental study of sheep scrapie: development and perspectives[END_REF]. Tg338 mice were imported from a specificpathogen-free (SPF) holding facility and bred in-house in a biosafety level 3 and SPF environment. Groups of 16 to 20 tg338 mice per isolate (8 to 10 weeks of age) were inoculated intracerebrally (ic) with 20 ml of 10% [w/v] brain tissue homogenates under isoflurane anesthesia. Mice were monitored for clinical signs of TSE at least twice per week, and were sacrificed when clinical signs were observed that indicated endstage TSE, or when a minimum survival time of 630 days had elapsed. At necropsy, the brain of every second animal was fixed in formalin for histopathology and immunohistochemistry. The remaining brains and the spleen and muscle tissues were snapfrozen in liquid nitrogen and stored at -20uC for western blot and ELISA analyses. Parallel samples were obtained from noninoculated mice of variable age that had served for breeding purposes (n = 11). A mouse was considered to be TSE-positive when PrP d was detected by Western blotting or immunohistochemistry in brain samples.

Ethics statement

All animal experiments were conducted according to the ''Ethical Principles and Guidelines for Experiments on Animals'' by the Swiss Academy of Medical Sciences and were approved by the Committee for Animal Experiments of the Canton of Berne (license #10/08).

Histopathology and lesion profiles

Mouse brains were fixed in 4% formaldehyde, decontaminated for 1 h in formic acid (98%) at room temperature, cut transversely into five pieces as suggested by Fraser and Dickinson [START_REF] Fraser | The sequential development of the brain lesion of scrapie in three strains of mice[END_REF], and embedded in paraffin. Four-mm thick tissue sections were stained with hematoxylin and eosin and vacuolar lesions were scored in ten defined grey matter areas on a scale from 0 to 5, and in four white matter areas on a scale from 0 to 3 [START_REF] Bruce | The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis[END_REF].

Immunohistochemistry for PrP d

The PrP d immunohistochemistry was performed on the same brain structures used for the lesion profiling, with a NEXEX robot (Ventana instruments) and monoclonal SAF84 antibody (SPI bio, A03208, 1:100) as described elsewhere [START_REF] Sigurdson | A molecular switch controls interspecies prion disease transmission in mice[END_REF], at the Institute of Neuropathology, University of Zu ¨rich. The PrP d deposition pattern was assessed at the NeuroCentre by microscopic examination of at least six mice per isolate, with the exception of isolate S4/RS, for which only one animal was available.

Western blotting

Whole brains from mice, and brain samples of donor sheep and goats, were homogenized and purified using a commercial TSE test kit (Bio-Rad, 355-1165) according to the manufacturer's instructions, but with the following modification: the kit-provided PK of unknown concentration was replaced by PK (Roche, 03115828001) at a final concentration of 50 mg/ml, which corresponds to 250 mg PK per gram wet tissue. The resulting pellets were resuspended in La ¨mmli sample buffer (Bio-Rad, 161-0737). PrP res fragments were separated by SDS-PAGE in hand-cast 16.5% tris-glycine gels or in pre-cast 4-20% gradient tris-glycine gels (Invitrogen, EC 6028), transferred to PVDF membranes (Bio-Rad, 162-0177), and blocked with 1% casein in PBS (Bio-Rad, 161-0783). Three PrP-specific mAbs were used: 9A2 ( 102 WNK 104 ) [START_REF] Langeveld | Rapid and discriminatory diagnosis of scrapie and BSE in retro-pharyngeal lymph nodes of sheep[END_REF], P4 ( 93 WGQGGSH 99 , rbiopharm, R8007), and Sha31 ( 148 YEDRYYRE 155 , Bio-Rad TeSeE Western Blot, 355-1169). The epitopes on the prion protein recognized by these mAbs have been schematically presented in a previous study [START_REF] Bouzalas | Distinct proteinase k-resistant prion protein fragment in goats with no signs of disease in a classical scrapie outbreak[END_REF]. Immunoreactivity was detected using a polyclonal rabbit anti-mouse HRP antibody (Dako, P0260), a chemiluminescent detection system (ECL-plus Amersham Biosciences), and a LAS 3000 TM imaging system (Fuji Film, Fuji Inc, Valhalla, NY). Protein bands were quantified and their molecular mass calculated with Quantity One software (Bio-Rad, Version 4.6.2) in comparison with a molecular mass standard (See-Blue, Invitrogen, LC5625).

Deglycosylation treatment

Following the resuspension of precipitated protein pellets in TD4215 (4% [w/v] SDS, 2% [v/v] b-mercaptoethanol, 192 mM Tris, 5% [w/v] sucrose), proteins were deglycosylated by incubation with the endoglycosidase PNGase F for 90 min at 37uC (New England Biolabs, P0704L) according to the manufacturer's instructions. Samples were mixed with an equal amount of La ¨mmli sample buffer and processed for western blotting as described above.

Statistical analyses

Survival time was defined as the number of days from inoculation to the animal's death. Animals that died in the first week following inoculation were excluded from the study. All statistical analyses were carried out using GraphPad's Prism software (Version 5.03). Survival times were compared between groups using the Log-rank Multiple-Comparison Test including the Bonferroni-correction (p,0.000758).

Supporting Information

Figure S1 Scheme of the neuroanatomical PrP d distribution in groups of tg338 mice inoculated with different scrapie isolates. (A) Presentation of the neuroanatomical structures investigated: cortex (cx), cingulum (cg), cerebral white matter (cw), corpus callosum (cc), caudate putamen (pu), septal nuclei (se), anterior commissure (ac), ventral pallidum (vp), piriform cortex (pi), hypothalamus (ht), lateral ventricle (lv), hippocampus (hi), hippocampal fissure (hf), habenula (ha), 3 rd ventricle (3v), thalamus (th), substantia nigra (sn), cerebral peduncle (cp), tractus opticus (ot), amygdala (am), superior colliculus (sc), nucleus brachium inferior colliculus (bi), pons (ps), aqueduct (aq), molecular layer (ml), granular layer (gl), cerebellar roof nuclei (rn), cerebellar white matter (wm), dorsal motor nucleus of the vagus nerve (dm), nucleus hypoglossus (hy), 4 th ventricle (4v), cerebellar peduncle (pe), spinal tract nucleus of trigeminal nerve (tn), spinal tract nucleus of trigeminal nerve (st), raphe (rf), pyramidal tract (pt). (B) PrP d is depicted for mild (dots) and severe (filled areas) deposits in the grey (red) and white matter (green) structures separately. Amyloid PrP d plaques were observed in the neuropil (w) as well as in apparent association with the cerebrospinal fluid space (q). (TIF)

1

  polymorphisms of the PRNP alleles are shown for position 136, 141,154 and 171 for the atypical scrapie isolates and for positions 136, 154 and 171 for the classical scrapie control. Alleles associated with relative susceptibility with atypical scrapie are indicated in bold. doi:10.1371/journal.pone.0027510.t001

Figure 1 .

 1 Figure 1. Survival of tg338 mice inoculated with different TSE isolates. Mice infected with Swiss atypical scrapie isolates (red) showed survival times similar to those infected with Nor98 (black), but these survival times were clearly shorter compared to the classical scrapie group (blue) and sham-inoculated mice (grey). doi:10.1371/journal.pone.0027510.g001

Figure 3 .

 3 Figure 3. Representative spongiform pathology in tg338 mouse brains. (A) Vacuolar lesions are evident in the hypothalamus, hippocampus, corpus callosum, and cerebral peduncle of TSE-inoculated and control mice. Classical scrapie-inoculated mice exhibit plaques in various brain structures such as the hippocampus (star). (B) Variability of spongiform lesions in the corpus callosum between mice inoculated with atypical scrapie isolates. (C) Cerebellum of a non-inoculated mouse showing mild (left) and a sham-inoculated mouse showing severe (right) vacuolar lesions. All microphotographs are 40X magnifications of hematoxylin and eosin-stained mouse brain sections. doi:10.1371/journal.pone.0027510.g003

Figure 4 .

 4 Figure 4. Neuroanatomical PrP d distribution in mice inoculated with different scrapie isolates. Microphotographs of representative mouse brain sections stained for PrP d by immunohistochemistry using antibody SAF84 are presented. For details on the anatomical structures, see Fig S1. doi:10.1371/journal.pone.0027510.g004

Figure 5 .Figure 6 .

 56 Figure 5. Localization and size of plaque-like PrP deposits in atypical and classical scrapie. In atypical scrapie-inoculated mice, relatively small plaques were observed in the neuropil (40X magnification), while the plaques in the classical scrapie-inoculated mice were larger, coalescing and always associated to the leptomeninges and the ependyma (20X magnification). PrP d immunoreactivity was detected using the mAb SAF84. Neuroanatomical structures: septal nuclei (se), caudate putamen (pu), lateral ventricle (lv) and thalamus (th). doi:10.1371/journal.pone.0027510.g005

Figure 7 .

 7 Figure 7. Molecular PrP res typing of atypical scrapie-affected sheep and goats. SDS-PAGE was performed using 4-20% gradient gels and blots were probed with mAb P4 (A) and Sha31 (B). Samples of a classical scrapie-affected sheep (cl) and a TSE-negative sheep (neg) served as controls. Molecular mass standards and tissue equivalents are indicated. doi:10.1371/journal.pone.0027510.g007

Figure

  Figure S2 Quantitative analysis of the PrP res phenotype in atypical scrapie affected tg338 mice. Molecular mass (yaxis, 6SD) and relative quantities of PrP res bands (numbers [%] at the right side of each dot) are depicted as means from at least five mice per isolates following SDS-PAGE using 4-20% gradient gels and monoclonal antibody P4. (TIF) Figure S3 Western blot banding patterns in the brains of sham-inoculated mice. (A) Tg338 mice inoculated with TSE-negative sheep brain show variable banding patterns after treatment with PK (50 mg/ml). Survival times (in days) of individual mice are indicated on the top. The picture on the left shows an atypical scrapie affected mouse-brain sample (S7/CS) for comparison. (B)The brain sample of a sham-inoculated mouse (610 days post-inoculation [dpi]) was treated with different concentrations of PK (indicated on top in mg/ml; BR: PK of the Bio-Rad TeSeE Western Blot test kit of unknown concentration). Upper bands of both mice are fully digested when using higher PK concentrations whereas an 8 kDa band in sham-inoculated mice are relatively PK-resistant. Controls: classical scrapie (cl), negative sheep (neg s). Molecular mass markers are indicated at each side, tissue equivalents loaded per well at the bottom. (TIF)

Table 1 .

 1 Small ruminant isolates, attack rates, and survival times in tg338 mice. #

			PrP d deposition intensity "		Attack rate	Mean survival time
	Inocula	Species	[Obx/Cbl/Cbr]	PRNP Genotype 1	[N d /N i ]	± SEM [days]
	Atypical scrapie
	G1/RS	Goat	1/-/-	A 136 L 141 H 154 Q 171 /ALHQ	17/17	23566.5
	G2/FS	Goat	1/1/3	ALHQ/ALHQ	14/14	20565.0
	S2/RS	Sheep	1/3/-	ALRR/ALRR	12/12	18864.8
	S3/RS	Sheep	1/3/-	ALRR/ALRR	16/16	22764.2
	S4/RS	Sheep	1/-/-	ALHQ/ALHQ	2/2*	189/256
	S5/FS	Sheep	1/1/3	ALRQ/ALHQ	16/16	21666.0
	S6/FS	Sheep	0/3/0	ALRR/ALRR	14/14	21366.0
	S7/CS	Sheep	1/3/3	AFRQ/AFRQ	14/14	21565.2
	Nor98	Sheep	1/3/3	ALHQ/ALHQ	16/16	22363.9
	Classical scrapie
	Classical scrapie	Sheep	3/2/3	V 136 R 154 Q 171 /A 136 R 154 Q 171	16/16	304617
	Negative
	Sham-inoculated	Sheep		n.a.	0/18	567622.4
	# abbreviations: Obx, obex; cbl, cerebellum; Cbr, cerebrum; n.a. not available; N i , number of mice inoculated; N d , number of mice diseased "

PLoS ONE | www.plosone.org

November 2011 | Volume 6 | Issue 11 | e27510

Acknowledgments

We would like to thank the Institute of Virology and Immunoprophylaxis (IVI), Mittelha ¨usern, Switzerland for providing containment animal facilities as well as technical assistance. Moreover, we are indebted to Michaela Gsponer, NeuroCentre, for excellent technical assistance as well as to Ingrid Kohler and Petra Schwarz, University of Zu ¨rich, for support in mouse inoculation, mouse husbandry, and automated IHC. Georg Krinke, AnaPath GmbH, Switzerland is acknowledged for expert advice on mouse brain histology. MAb 9A2 was a gift from Jan Langeveld, Central Veterinary Institute of Wageningen UR, the Netherlands. Excellent editing service was provided by Alison E. Mungenast.

This study was funded by the Swiss Federal Veterinary Office (grant #1.08.13.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions

Conceived and designed the experiments: AO AZ TS. Performed the experiments: DRG. Analyzed the data: DRG AO TS. Contributed reagents/materials/analysis tools: SLB HL. Wrote the paper: DRG TS.


K K Aupart

Steenkeste

S Daddi Oubekka

R Briandet

M.-P Fontaine-Aupart

K Steenkeste
email: karine.steenkeste@u-psud.fr.

Correlative Time-Resolved Fluorescence Microscopy To Assess Antibiotic Diffusion-Reaction in Biofilms

HAL is a

B iofilms are composed of microorganisms entrapped in a hy- drated matrix of organic exopolymeric substances (EPS) produced by the cells themselves. EPS are mainly composed of polysaccharides, proteins, nucleic acids, lipids, and heteropolymers that are interlaced by a vast number of weak interactions (hydrogen bonds, hydrophobic, electrostatic, van der Waals interactions, divalent cation bridges, etc.) giving the three-dimensional assemblage a viscoelastic nature [START_REF] Flemming | The biofilm matrix[END_REF]. Such a reactive molecular environment pointed to the proposal that this biomass could act as a diffusion barrier that lessens the antimicrobial bioavailability and thus be a contributing factor to biofilm antibiotic tolerance. This is the case of tobramycin, a polycationic aminoglycoside that interacts electrostatically with the polyanionic alginate matrix of Pseudomonas aeruginosa biofilms [START_REF] Nichols | Inhibition of tobramycin diffusion by binding to alginate[END_REF]. In addition, antibiotic sorption may be highly dependent on microenvironment heterogeneity (dense clusters of polysaccharides, DNA resulting from cell lysis, etc.) that can also induce heterogeneity of the antibiotic irrigation. The cells are thus locally exposed to various concentrations of antibiotics, leading to some subinhibitory concentrations of the antibiotic through the biofilms.

Fluorescence microscopy represents an essential and noninvasive method to assess antimicrobial dynamics and reactivity in biofilms, contributing to our understanding of what makes bacteria resistant in these biostructures [START_REF] Bridier | Resistance of bacterial biofilms to disinfectants: a review[END_REF][START_REF] Bridier | Deciphering biofilm structure and reactivity by multiscale time-resolved fluorescence analysis[END_REF][START_REF] Davison | Spatial and temporal patterns of biocide action against Staphylococcus epidermidis biofilms[END_REF][START_REF] Takenaka | Direct visualization of spatial and temporal patterns of antimicrobial action within model oral biofilms[END_REF]. In this respect, confocal laser scanning microscopy (CLSM) has been shown to be a powerful tool to analyze antimicrobial actions indirectly by a time course (hour-to-day scale) visualization of live and dead cells through the biofilm structure [START_REF] Bridier | Dynamics of the action of biocides in Pseudomonas aeruginosa biofilms[END_REF][START_REF] Hope | Analysis of the effects of chlorhexidine on oral biofilm vitality and structure based on viability profiling and an indicator of membrane integrity[END_REF][START_REF] Takenaka | Direct visualization of spatial and temporal patterns of antimicrobial action within model oral biofilms[END_REF][START_REF] Zheng | Growth limitation of Staphylococcus epidermidis in biofilms contributes to rifampicin tolerance[END_REF]. However, this approach is unlikely to allow distinguishing survivor cells that were not reached by the active molecules (diffusion-reaction limitation through the matrix) from survivors with resistant physiology (biofilm "phenotype").

More recently, the emergence of fluorescently tagged antimicrobials, including antibiotics, allowed their direct tracking (second-to-minute scale) through biofilms by time-lapse microscopy [START_REF] Jefferson | Use of confocal microscopy to analyze the rate of vancomycin penetration through Staphylococcus aureus biofilms[END_REF][START_REF] Stone | Tetracycline rapidly reaches all the constituent cells of uropathogenic Escherichia coli biofilms[END_REF]. The available studies highlight various behaviors depending on the biofilm models and/or the antibiotics considered. For example, it was observed that tetracycline and daptomycin quickly (i.e., within a few minutes) make their way into Escherichia coli and Staphylococcus epidermidis biofilms, respectively [START_REF] Stewart | Daptomycin rapidly penetrates a Staphylococcus epidermidis biofilm[END_REF][START_REF] Stone | Tetracycline rapidly reaches all the constituent cells of uropathogenic Escherichia coli biofilms[END_REF], whereas it takes about 1 h for vancomycin to diffuse through a mucoid model of Staphylococcus aureus biofilm [START_REF] Jefferson | Use of confocal microscopy to analyze the rate of vancomycin penetration through Staphylococcus aureus biofilms[END_REF]. From these studies, it is evident that no consensus has been reached yet on the role of the EPS matrix of biofilms as a barrier to the diffusionreaction of antibiotics.

Although time-lapse imaging represents an essential versatile tool, visualizing in real time the penetration of the antibiotic through the biofilm depth, the method is limited in its temporal resolution giving only access to initial diffusive rates. No further measurements are available when the antimicrobials have reached equilibrium inside the biomatrix, although it is important to follow their reactivity on the time scale of antibiotic exposure (several hours to days). To overcome this limitation, we took advantage of the large potential offered by advanced CLSM methods, including fluorescence recovery after photobleaching (FRAP) [START_REF] Oubekka | Image-based fluorescence recovery after photobleaching (FRAP) to dissect vancomycin diffusion-reaction processes in Staphylococcus aureus biofilms[END_REF][START_REF] Waharte | Diffusion measurements inside biofilms by image-based fluorescence recovery after photobleaching (FRAP) analysis with a commercial confocal laser scanning microscope[END_REF], fluorescence correlation spectroscopy (FCS) [START_REF] Briandet | Fluorescence correlation spectroscopy to study diffusion and reaction of bacteriophages inside biofilms[END_REF][START_REF] Guiot | Heterogeneity of diffusion inside microbial biofilms determined by fluorescence correlation spectroscopy under two-photon excitation[END_REF][START_REF] Habimana | Diffusion of nanoparticles in biofilms is altered by bacterial cell wall hydrophobicity[END_REF], and fluorescence lifetime imaging (FLIM) [START_REF] Bridier | Deciphering biofilm structure and reactivity by multiscale time-resolved fluorescence analysis[END_REF][START_REF] Guiot | Heterogeneity of diffusion inside microbial biofilms determined by fluorescence correlation spectroscopy under two-photon excitation[END_REF][START_REF] Neu | Advanced imaging techniques for assessment of structure, composition and function in biofilm systems[END_REF]. These methods have not only allowed significant advances owing to their performance in terms of spatial resolution (submicrometric) and sensitivity (nanomolar-to-micromolar therapeutic antibiotic concentrations), but they have also provided noninvasive conditions of observations consistent with studies on biofilms after several hours of contact with the antibiotics. They allow discriminating free diffusion from constrained diffusion of the antibiotics and hence quantifying their interaction with biofilm components.

The purpose of the present study was to highlight the strength of such correlative time-resolved fluorescence microscopy approaches in order to assess and understand the diffusion-reaction of fluorescently labeled vancomycin within S. aureus biofilms of two different human isolates.

MATERIALS AND METHODS

Bacterial strain, media, and antibiotics. Two S. aureus strains (ATCC 6538 and ATCC 27217) and one P. aeruginosa strain (ATCC 15442) were used in the present study. The stock cultures were kept at Ϫ80°C in tryptic soy broth (TSB; BD-Difco, France) containing 20% (vol/vol) glycerol.

Prior to each experiment, the frozen cells were subcultured twice in TSB. Bacterial growth and experiments were both conducted at 30°C. The fluorescently labeled antibiotic BODIPY-vancomycin and free BODIPY (4,4difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid) were purchased from Invitrogen (Cergy-Pontoise, France). The lyophilysates were dissolved in sterile Milli-Q water for BODIPY-vancomycin and in dimethyl sulfoxide for BODIPY to obtain stock solutions of 69 and 34.5 mM, respectively. These stock solutions, kept at Ϫ20°C before use, were then diluted to working concentrations: 345 nM for FCS, 6.9 M for FLIM, and 69 M and 34.5 mM for FRAP. Biofilm preparation. For the preparation of S. aureus biofilms, 500-l portions of an overnight subculture adjusted to an optical density at 600 nm of 0.03 (corresponding to ϳ10 7 CFU/ml) were added to 4-well sterile microscopic chambers (Lab-Tek; Nunc/Thermo Scientific, France), or 250-l portions were added to 96-well microplates (Clear; Greiner Bio-One, France) under humidified air conditions. After a 1.5-h adhesion period at 30°C, the wells were rinsed with 150 mM NaCl in order to eliminate nonadherent cells, refilled with sterile TSB, and then incubated for 24 h at 30°C to allow biofilm growth. Since TSB contains traces of fluorescent compounds, the biofilms were rinsed with 150 mM NaCl and refilled with nonfluorescent RPMI 1640 medium (Gibco/Invitrogen, France). Three-day-old P. aeruginosa biofilms were prepared according to S. aureus biofilm protocol but using M9 minimal growth medium (Difco, France) that was replaced with fresh medium every day. Examples of the distribution of cells, EPS, and interstitial voids after a 24-h biofilm growth period are provided in Fig. S1 in the supplemental material. Visualization of antibiotic cell wall binding. The antibiotic cell binding was visualized as described by Gilbert et al. [START_REF] Gilbert | Single-molecule force spectroscopy and imaging of the vancomycin/D-Ala-D-Ala interaction[END_REF]. Briefly, BODIPYvancomycin was mixed with an equal amount of unlabeled vancomycin, giving a final concentration of 3 g/ml, and the mixture was added to S. aureus cells from exponentially growing cultures. After a 30-min incubation period at 30°C, the cells were deposited on a polylysine-coated slide (Kindler GmbH; VWR, France) and then visualized by fluorescence microscopy. In addition, the visualization of the nascent peptidoglycan was obtained according to the protocol of Pinho and Errington [START_REF] Pinho | Dispersed mode of Staphylococcus aureus cell wall synthesis in the absence of the division machinery[END_REF].

Visualization of antibiotic penetration. BODIPY-vancomycin diffusive penetration through our biofilm models was measured by time-lapse microscopy using a Leica SP2 AOBS confocal laser scanning microscope (Leica Microsystems, France), implemented at the MIMA2 microscopy platform (Massy). For these experiments, the labeled antibiotic was excited with a continuous argon laser line at 488 nm through a ϫ63 oil immersion objective, and the emitted fluorescence was recorded within the range 500 to 650 nm. The focal plane inside the biofilms (thickness, ϳ30 m) was set ϳ5 m above the glass surface. An x-y time series was then initiated in which an image of 512 ϫ 128 pixels corresponding to ϳ40 ϫ 10 m 2 was collected every 3 s. The time series was recorded for 10 min. Images were analyzed using Leica software (Lite; Leica Microsystems). BODIPY and BODIPY-vancomycin solutions were added very gently to the medium over the biofilm; transmission imaging ascertains that no structural alteration of the biofilm occurred during this process.

The corresponding diffusive penetration coefficients (D p ) through the biofilms were determined according to the relationship described by Stewart for flat structures [START_REF] Stewart | Diffusion in biofilms[END_REF]:

D p ϭ 1.03 ϫ L 2 ⁄ t 90 ( 1
)
where L is the biofilm thickness and t 90 is the time required to attain 90% of the equilibrium staining intensity at the deeper layers of the biofilm. Time-resolved fluorescence microscopy. All time-resolved fluorescence measurements were obtained on the same confocal Leica SP5 microscope (Leica Microsystems; implemented at the Centre de Photonique Biomédicale of Orsay) equipped with a ϫ63 high numerical aperture (1.4) oil immersion objective and coupled with either continuous lasers for FCS and FRAP acquisitions or a femtosecond titanium-sapphire laser (Chameleon-XR; Coherent, USA) running at a 80-MHz repetition rate and delivering pulses of 150 fs for fluorescence decay time and FLIM measurements.

FRAP. The principle of FRAP is based on a brief excitation of fluorescent molecules by a high-intensity laser pulse spatially confined to the volume defined by the confocal microscope objective (for single-spot FRAP) or, in a user-defined region, to quench irreversibly their fluorescence (photobleaching). Fluorescence recovery in the photobleached area is then observed as a function of time.

In the present study, an image-based FRAP protocol was applied that can be readily applied by anyone familiar with a confocal laser scanning microscope and is well adapted to study in situ local molecular diffusion with accuracy. This protocol includes (i) the image acquisition of photobleached areas and (ii) a kymogram representation to control the bacterial movement that may lead to an incorrect estimation of the molecular diffusion coefficient [START_REF] Waharte | Diffusion measurements inside biofilms by image-based fluorescence recovery after photobleaching (FRAP) analysis with a commercial confocal laser scanning microscope[END_REF]. Kymograms are two-dimensional graphs showing fluorescence intensity fluctuations over time along a chosen trajectory. Such representation is essential in order to discard distorted acquisitions.

The image-based FRAP protocol has been explained in detail elsewhere [START_REF] Oubekka | Image-based fluorescence recovery after photobleaching (FRAP) to dissect vancomycin diffusion-reaction processes in Staphylococcus aureus biofilms[END_REF][START_REF] Waharte | Diffusion measurements inside biofilms by image-based fluorescence recovery after photobleaching (FRAP) analysis with a commercial confocal laser scanning microscope[END_REF]. Briefly, the fluorescence intensity image size was fixed to 512 ϫ 128 pixels with a 80-nm pixel size in order to ensure usable spatial information on the biofilm (ϳ40 ϫ 10 m 2 ) and recorded using 16-bit resolution to improve the image analysis. The line scan rate frequency was fixed at 1,400 Hz, which corresponds to a total time between frames of ϳ265 ms. The full widths at half-maximum in the x and y directions and the z direction (i.e., along the optical axis) of the bleached profiles were 0.8 and 14 m, respectively, as determined previously [START_REF] Waharte | Diffusion measurements inside biofilms by image-based fluorescence recovery after photobleaching (FRAP) analysis with a commercial confocal laser scanning microscope[END_REF].

Each FRAP experiment started with the acquisition of 50 image scans at 3% of laser maximum intensity (which was measured to be ϳ7 W at the object level) and followed by a single bleached spot of 100 ms at 100% laser intensity. A series of 300 single-section images were then collected with the laser power attenuated to its initial value (3% of the bleach intensity); the first image was recorded 365 ms after the beginning of bleaching. Under these image acquisition conditions, FRAP measurements could be acquired in 74 s, and this ensured bacterial viability as controlled with a live-dead staining.

The fluorescence recovery curves here reported were analyzed using the adapted mathematical model of Braga et al. [START_REF] Braga | Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes[END_REF] as previously detailed [START_REF] Waharte | Diffusion measurements inside biofilms by image-based fluorescence recovery after photobleaching (FRAP) analysis with a commercial confocal laser scanning microscope[END_REF]. It should be noted that a two-dimensional diffusion was considered in view of the axial (14-m) and lateral (0.8-m) extents of the photobleaching pattern; diffusion along the axial/vertical axis can be neglected.

FCS. Fluorescence correlation spectroscopy (FCS) is based on monitoring the emission intensity fluctuations due to a small number of fluorophores (until probes concentrations of few hundreds nanomolar) passing through the confocal excitation volume. These fluctuations can be quantified in their amplitudes and durations by temporally autocorrelating the recorded intensity signals. In the absence of chemical reaction or other dynamic processes, these temporal fluctuations in intensity can be attributed solely to the translational diffusion of the fluorescent probes.

Our FCS measurements were performed point by point using the 488-nm line of a continuous Ar laser. The emission output was focused on a PicoHarp 300 module (PicoQuant, Germany) equipped with a singlephoton avalanche diode detector (time resolution of 250 ps). The emitted fluorescence was recorded within a range of 500 to 650 nm. Data acquisition was performed using SymPhoTime software (PicoQuant), which computes online the correlation function of fluorescence fluctuations. To measure the translational diffusion time with 10 to 20% accuracy, fluo-rescence temporal signals in water were collected in runs of 60 s (number of iterations, 5 to 10). In the biofilms, acquisition time was increased to 120 s, and each experiment was repeated 10 times at ϳ15 randomly selected locations (to account for biofilm heterogeneity). The data were analyzed using both SymPhoTime software (PicoQuant) and an in-house Matlab routine, which enabled (i) the selection and averaging of chosen successive acquisitions, (ii) the adaptation of different diffusion models to analyze the autocorrelation function, and (iii) the calculation of associated standard deviations. The incident laser energy was attenuated to less than 1 mW at the sample in order to reduce the risk of photobleaching.

In these experiments, we assumed that the excitation intensity profile could be approximated using a three-dimensional Gaussian distribution and that no additional "blinking" dynamics of the fluorophore occurred other than its diffusion through the excitation volume; then each fluorescence autocorrelation [g()] curve was fitted using a two-component free Brownian motion model:

g() ϭ 1 ͙ 8N ͫ a ͩ 1 1 ϩ ( ⁄ 1 ) ͪͩ 1 1 ϩ ( 0 ⁄ z 0 ) 2 ( ⁄ 1 ) ͪ 1⁄2 ϩ (1 Ϫ a) ͩ 1 1 ϩ ( ⁄ 2 ) ͪͩ 1 1 ϩ ( 0 ⁄ z 0 ) 2 ( ⁄ 2 ) ͪ 1⁄2 ͬ (2)
where N is the number of fluorescent molecules in the excitation volume; ␣ and "(1 Ϫ ␣)" are the fractions of the molar concentrations of the two fluorescent diffusive species, 1 and 2 are their respective translational diffusion times, and 0 and z 0 are the radial and axial radii of the confocal volume. These were determined by previous calibration experiments ( 0 ϭ 0.25 Ϯ 0.04 m and z 0 ϭ 0.84 Ϯ 0.1 m) and fixed throughout the fittings. In the present study, this two-component model allowed us to discriminate the fast-diffusing fluorescent molecules of antibiotics from the slower ones, which are either autoaggregates or complexed with the biofilm components. The diffusion coefficient (D) could be related to the translational diffusion time i using the following equation:

D ϭ 0 2 ⁄ 4 i (3)
All of the FCS data reported here are averages of at least 10 independent sample preparations. Fluorescence lifetime and FLIM measurements. Fluorescence intensity is dependent on fluorophore concentration and on environmental conditions and thus can be difficult to understand or interpret. Fluorescence decay time measurements allowed us to address this problem. Fluorescence decay time corresponds to the average time a fluorophore remains in the excited state after excitation. The fluorescence lifetime is an intrinsic characteristic of the fluorophore, independent on its concentration, but may locally vary depending on the reactivity of the fluorophore with the biological environment. It is thus possible to build fluorescence lifetime images that allow assessment of the reactivity of a fluorophore at the molecular level throughout a three-dimensional biological structure such as a biofilm.

In the present study, the BODIPY-labeled samples were biphotonically excited (two-photon excitation) at 950 nm. The fluorescence signal was collected by using a PicoHarp 300 device based on time-correlated single-photon counting method. An 800-nm short-pass emission filter was used to remove any residual laser light, and the emitted fluorescence was recorded within a range of 500 to 650 nm.

The observed time-resolved decays were deconvoluted with the instrumental response function obtained on a picric acid solution. It was possible here to fit all of the fluorescence decays, with a single exponential giving the fluorescence lifetime of the sample with a 100-ps time resolution [START_REF] Valeur | Molecular fluorescence: principles and applications[END_REF]. To obtain two-dimensional FLIM, the laser beam was scanned on the cell surface at 400 Hz, which gave an acquisition mean time of ϳ10 min to cover a field of view of 50 by 50 m 2 (64 ϫ 64 pixels).

RESULTS

Assessing BODIPY-vancomycin interaction with planktonic S. aureus bacteria. The specific binding of BODIPY-vancomycin to planktonic S. aureus bacteria was determined by using both FCS and time-resolved fluorescence emission.

Before carrying out studies in the presence of bacteria, FCS measurements were performed using the free BODIPY and BODIPY-vancomycin probes in aqueous solution (water viscosity ϭ 0.96 cF at 295 K). Typical experimental fluorescence correlation curves are presented in Fig. 1a, showing a reproducible shift of BODIPY-vancomycin curve compared to that of free BODIPY. The diffusion times obtained by fitting the experimental curves (equation 2) and the corresponding diffusion coefficient values calculated using equation 3 are summarized in Table 1. The slight difference between the major diffusion coefficient (D 1 ) of BODIPY-vancomycin and the one of free BODIPY in the data measured was in line with the influence of the probe size on mobility. The minor diffusion coefficient D 2 of BODIPY-vancomycin likely corresponds to molecular aggregates.

We checked that the remaining autofluorescence of planktonic S. aureus bacteria was high enough to create an additional correlation signal as illustrated in Fig. 1b. The corresponding time range of bacteria diffusion was ca. 30 to 100 ms, depending on the presence of cell aggregates. In the presence of free BODIPY, the fluorescence correlation curve analysis using equation 2 corresponds to a combination of the diffusion coefficients for BODIPY and planktonic bacteria (Fig. 1a, inset, and Table 1). Because of this bacterial contribution, some dispersion in the fast BODIPY diffusion time was observed compared to water. However, it can be ascertained that the probe diffusion remains unchanged in the presence of bacteria.

By contrast, the correlation curves measured after adding BODIPY-vancomycin to a bacterial suspension could be superposed on those obtained with planktonic bacteria in solution (Fig. 1b and Table 1). Furthermore, a much more intense fluorescence count rate burst was recorded in the presence of BODIPY-vanco-mycin (Fig. 1c) compared to planktonic bacteria that indicates the presence of the antibiotic in the bacterial suspension. Together, these results demonstrate that BODIPY-vancomycin interacts with planktonic bacteria from the vancomycin part of the molecule, which is confirmed by time-resolved fluorescence emission.

In the absence of bacteria, the fluorescence decay of BODIPYvancomycin could be superposed on that of free BODIPY. It is monoexponential, corresponding to a fluorescence lifetime of 5.7 Ϯ 0.2 ns (Table 1 and Fig. 2a), which is in good agreement with a previous report [START_REF] Karolin | Fluorescence and absorption spectroscopic properties of dipyrrometheneboron difluoride (BODIPY) derivatives in liquids, lipid membranes, and proteins[END_REF]. The presence of bacteria does not affect the dynamic fluorescence properties of BODIPY. Combining this result with FCS data suggests the absence of strong interaction between the fluorescent probe and the bacterial cell wall. In contrast, the BODIPY-vancomycin fluorescence lifetime is shortened to 4.5 Ϯ 0.2 ns in the presence of bacteria (Table 1 and Fig. 2). Such a variation in the fluorescence decay time reflects a change in the vicinity of the fluorophore, which is consistent with an interaction of the vancomycin part of the labeled antibiotic with the bacterial surface.

In the light of these data, we examined the possibility of visualizing the cellular binding pattern of the fluorescent conjugated antibiotic by fluorescence microscopy using the protocol described in Materials and Methods. Figure 3a shows that BODIPYvancomycin was not distributed evenly around the planktonic cell wall but preferentially to the division septum sites upon cell division (Fig. 3b). This is in good agreement with the reported specificity and affinity of the antibiotic to bind preferentially to the terminal D-Ala-D-Ala of nascent peptidoglycan at the cell septum but also to the dipeptide present on the surface of the bacteria [START_REF] Gilbert | Single-molecule force spectroscopy and imaging of the vancomycin/D-Ala-D-Ala interaction[END_REF][START_REF] Pinho | Dispersed mode of Staphylococcus aureus cell wall synthesis in the absence of the division machinery[END_REF]. Similar fluorescence imaging was also performed in the presence of free BODIPY: neither bacterial nor BODIPY fluorescence was detected, confirming that the fluorophore did not interact with the S. aureus cell wall.

BODIPY-vancomycin diffusion-reaction inside S. aureus biofilms. (i) Time-lapse imaging. Visualization of both BODIPY

and BODIPY-vancomycin penetration through S. aureus biofilm depths (thickness, ϳ30 m) was performed. The fluorescence signals at the deeper layers of the biofilm (biofilm slide adjacent to the coverslip) were measured within 2 to 3 min for S. aureus ATCC 6538 and within ϳ8 min for S. aureus ATCC 27217 (Fig. 4 and see Movie S1 in the supplemental material). The fluorescence inten- sity reached a plateau that remained steady. The equilibrium distribution of the labeled antibiotic through each S. aureus strain is illustrated in Fig. 4c andd. We observed that the antibiotic labeled preferentially bacterial cell walls through all depths of the biofilms. By comparison, maximal fluorescence of free BODIPY occurred within the time taken to obtain an x-y plane image (Ͻ3 s). The corresponding D p values obtained from equation 1 are listed in Table 2. Diffusion of free BODIPY was too fast to be quantified by time-lapse confocal imaging.

(ii) FRAP imaging. Our group has recently developed an image-based FRAP protocol to improve the accuracy of FRAP measurements inside biofilms [START_REF] Oubekka | Image-based fluorescence recovery after photobleaching (FRAP) to dissect vancomycin diffusion-reaction processes in Staphylococcus aureus biofilms[END_REF][START_REF] Waharte | Diffusion measurements inside biofilms by image-based fluorescence recovery after photobleaching (FRAP) analysis with a commercial confocal laser scanning microscope[END_REF]. In particular, we have introduced a kymogram representation (illustrated in Fig. 5a andd) to check biofilm stability during fluorescence recovery curve acquisitions and to validate the data.

FRAP experiments (image acquisition, fluorescence recovery curve) of BODIPY and BODIPY-vancomycin in each type and different parts of S. aureus biofilms were performed (Fig. 5b ande). Over the time range of our observation (74 s), complete fluorescence recovery of BODIPY was observed (Fig. 5c). In contrast, only partial recovery of BODIPY-vancomycin was measured inside each strain of S. aureus biofilm corresponding to a mobile fraction, respectively, of ca. 60% Ϯ 10% in S. aureus ATCC 6538 biofilm and ranging from 20 to 60% in S. aureus ATCC 27217 biofilm (Fig. 5f) depending on the local EPS nature: FRAP measurements acquired in extracellular DNA clusters (corresponding to the red zones in Fig. S1 in the supplemental material) reveal much lower mobile fractions. A control experiment showed that without the photobleaching phase, the fluorescence signal is stable during the acquisition time. The local diffusion coefficients at equilibrium (D) determined using the adapted mathematical model of Braga described in Materials and Methods are summarized in Table 2. They are of the same order of magnitude as D p estimated from time-lapse confocal imaging analysis.

Otherwise, the image sequences acquired during FRAP measurements (Fig. 5b ande) reinforce the privileged location of the antibiotic on the bacterial cell surface, whereas free BODIPY localized in the intercellular space.

(iii) FLIM measurements. A typical fluorescence lifetime image of BODIPY-vancomycin in S. aureus biofilm is reported in Fig. 2b. On an x-y plane, the fluorescence lifetime appears homogeneous over the entire surface. The corresponding fluorescence decay times acquired by collecting all of the photons in an x-y plane gave an average lifetime f of 5.0 Ϯ 0.2 ns. That can be correlated with 50% of BODIPY-vancomycin molecules in their free form ( f ϭ 5.7 ns) and 50% in their bound form to S. aureus biofilm components, as determined in the presence of planktonic bacteria ( f ϭ 4.5 ns).

(iv) FCS measurements. FCS measurements were also performed in different areas of S. aureus biofilms (at the biofilm base and surface and in the bulk biofilm). In the absence of BODIPY-vancomycin, only small fluorescence intensity fluctuations around a low mean value are observed (Fig. 6a,inset). The analysis of corresponding autocorrelation curves leads to a diffusion time of a few hundred milliseconds (Fig. 6b). This value, which was higher than that obtained for planktonic cells, is consistent with the constricted movement of bacteria embedded in an exopolymeric matrix, which is more viscous than a culture medium [START_REF] Braga | Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes[END_REF]. We also checked to determine whether free BODIPY has similar diffusion properties inside the biofilm as in an aqueous environment, confirming that no strong interaction occurs between the fluorophore and bacteria and/or the EPS matrix (Fig. 6b,insert).

For S. aureus biofilms treated with BODIPY-vancomycin, the recorded fluorescence time trace (Fig. 6a) shows a signifi-cant increase in the fluorescence intensity fluctuations compared to the time trace obtained in the absence of the antibiotic. Whatever the area considered, diffusion times of the same order of magnitude as in untreated biofilms were obtained corresponding to that of the global movement of the biofilm (Fig. 6a). This suggests that, at the nanomolar concentration used in the FCS measurements, there was a total interaction of vancomycin with S. aureus biomass.

In order to test whether the inhibition of free antibiotic diffusion could be attributed to nonspecific interactions with the biomass, FCS experiments were extended to P. aeruginosa biofilm incubated with BODIPY-vancomycin. This Gram-negative bacterium is known to be insensitive to the antibiotic due to the absence of the D-Ala-D-Ala targets on the outer cell envelope. Figure 6c shows a typical autocorrelation curve, obtained over several horizontal scans and at different depths within low, dense zones of the biofilm matrix (see Fig. S1c in the supplemental material). The corresponding BODIPY-vancomycin diffusion time 1 is 67 Ϯ 25 s (D ϭ 224 Ϯ 80 m2 s Ϫ1 ), which is close to the value obtained for unreactive BODIPY in S. aureus biofilm). This shows that the labeled antibiotic freely diffuses through a biofilm composed of cells devoid of antibiotic targets.

DISCUSSION

Medicine today is faced with the consideration of infections involving biofilms on both the surface and the internal tissues of the host or on invasive devices such as catheters and implants [START_REF] Bryers | Medical biofilms[END_REF]. The presence of biofilms (versus planktonic cells) during infection is not diagnosed by current methods of medical bacteriology (except in few cases for superficial infections), and thus all infections are treated the same way. However, experimental evidence has accumulated showing that the biofilm can shield the action of antimi-crobials and of the immune system [START_REF] Fux | Survival strategies of infectious biofilms[END_REF]. Hence, antibiotics with high performance against planktonic exponential-growth-phase bacteria in vitro may be less successful in clearing biofilm infections in vivo.

Understanding the mechanisms of biofilm resistance to antimicrobials, especially antibiotics, has become a challenge for the medical community. This is especially important at a time when bacterial strains resistant to any form of conventional antibiotherapy are emerging, as is the case for S. aureus [START_REF] Hiramatsu | The emergence and evolution of methicillin-resistant Staphylococcus aureus[END_REF].

It is now well established that horizontal gene transfers, and in particular those related to antibiotic resistance, occur at a much higher frequency between cells in a biofilm than between their planktonic counterparts [START_REF] Høiby | Antibiotic resistance of bacterial biofilms[END_REF]. However, in addition to these acquired traits of genetic resistance, biofilm spatial organization may also be the cause of high antibiotic tolerance [START_REF] Høiby | Antibiotic resistance of bacterial biofilms[END_REF]. The associated mechanisms may involve specific cell physiology and the matrix of the biofilm that could act as a barrier to the diffusionreaction of the antibiotics by retarding its penetration and/or restricting its bioavailability.

We sought here to dissect noninvasively the action of vancomycin on S. aureus biofilms using a set of advanced dynamic fluorescence imaging methods, including not only confocal timelapse imaging, which is already widely used, but also FLIM, FRAP, and FCS methods.

Is the exopolymeric matrix of S. aureus biofilms an obstacle to the diffusion-reaction of vancomycin? The only results available in the literature on the mobility of BODIPY-vancomycin inside S. aureus biofilms were reported by Jefferson et al. [START_REF] Jefferson | Use of confocal microscopy to analyze the rate of vancomycin penetration through Staphylococcus aureus biofilms[END_REF]. These researchers observed a low rate of penetration of the antibiotic through their highly mucoid matrix (full biofilm irrigation with vancomycin can take Ͼ1 h). It was then hypothesized that a gradual cell exposure to the antibiotic could allow bacteria to undergo stress induced by metabolic or transcriptional changes that could increase their tolerance toward the antibiotic [START_REF] Jefferson | Use of confocal microscopy to analyze the rate of vancomycin penetration through Staphylococcus aureus biofilms[END_REF].

Our data obtained by time-lapse microscopy and fluorescence imaging demonstrated that BODIPY-vancomycin penetrates to the deepest layers (ϳ30 m) of our S. aureus biofilms within minutes (2 to 3 min for ATCC 6538 biofilm and 8 min for ATCC 27217 biofilm) to reach preferentially the cell walls of the embedded bacteria (Fig. 4c andd). Remarkably, no measurable activity of the antibiotic on biofilm cell viability was recorded either by time-lapse imaging using the fluorescent live-dead staining or by conventional plating on agar. It was also shown that the BO-DIPY labeling of the antibiotics does not modify its antibacterial activity against both the S. aureus strains (data not shown). This fast irrigation by the antibiotic of the whole biofilm structure excludes bacterial stress response. The findings of Jefferson et al. may be related to the particular density of the EPS matrix due to poly-N-acetylglucosamine (PNAG) overproduction by the MN8m strain they used.

In the present study, we showed that fluorescently tagged vancomycin penetrates each S. aureus biofilm (time-lapse measurements) and further diffuses through the biomass (FRAP measurements) with similar diffusion coefficients. However, the relative values (i.e., the ratio between the diffusion coefficients measured in a biofilm and in water) of BODIPY-vancomycin in the S. aureus biofilms (ca. 0.01 to 0.03) do not range over the same scale as that commonly obtained for molecules of comparable molecular weight (0.1 to 0.35) [START_REF] Rani | Rapid diffusion of fluorescent tracers into Staphylococcus epidermidis biofilms visualized by time lapse microscopy[END_REF]. Furthermore, both FRAP and FLIM results reveal that at equilibrium (after a lag phase of about 30 min) approximately half of the antibiotic molecules are immobilized inside the biofilms.

The weak change in viscosity (ratio of ϳ1.5) (2) between water and biofilm cannot be the only explanation for this diffusion lim- S1c in the supplemental material). The fit of the curves (straight lines) was obtained using equation 2.

itation. Electrostatic interaction must also be considered due to the weak cationic charge of vancomycin (ϩ0.7) (31), the globally negative charge of the cell surfaces at neutral pH [START_REF] Gross | Key role of teichoic acid net charge in Staphylococcus aureus colonization of artificial surfaces[END_REF][START_REF] Klodzinska | Differentiation of Staphylococcus aureus strains by CE, zeta potential and coagulase gene polymorphism[END_REF], and to a lesser extent the charged components of the EPS matrix. Indeed, it was previously reported by FCS measurements that electrostatic attraction between the probe and the biofilm resulted in a reduced diffusion of the probes [START_REF] Guiot | Heterogeneity of diffusion inside microbial biofilms determined by fluorescence correlation spectroscopy under two-photon excitation[END_REF][START_REF] Zhang | Quantifying diffusion in a biofilm of Streptococcus mutans[END_REF]. Nonetheless, if such an electrostatic attractive effect governed the molecular mobility of the antibiotic, it would have also been observed in P. aeruginosa biofilm, which also has anionic charges on its cell walls and possibly on its extracellular polysaccharides [START_REF] Shephard | Mechanisms of cation exchange by Pseudomonas aeruginosa PAO1 and PAO1 wbpL, a strain with a truncated lipopolysaccharide[END_REF]; however, this was not the case.

More reasonably, the slow vancomycin diffusion through the S. aureus biofilms was likely due to specific adsorption of the antibiotic to the biofilm components. As mentioned above, the bacterial cell walls appeared as hot spots on fluorescence images, revealing the specific interaction of the antibiotic with its target. Nevertheless, some EPS components (proteins, polysaccharides, extracellular DNA, divalent ions, etc.) may also be expected to play a part in vancomycin immobilization inside the biomass. FRAP experiments in the extracellular DNA pockets of the ATCC 27217 biofilm matrix support this hypothesis. Indeed, in these specific zones, much higher immobilized fractions of the antibiotic are measured (60% versus ϳ40%), a finding which is in good agreement with the reported ability of vancomycin to intercalate into DNA molecules [START_REF] Vijan | The interaction of vancomycin with DNA[END_REF].

Is vancomycin bioavailability an obstacle to its activity inside S. aureus biofilms? Failure for some antibiotics to be active on cell viability in biofilms is often attributed to the failure of the drug to penetrate the biostructure. Our study is an additional contribution supporting that biofilm matrix is not an absolute physical barrier to the penetration of molecules at the size of antibiotics. However, sorption to, or reaction with, biofilm components may reduce antibiotic bioavailability and also be put forward to explain the decrease in antibiotic efficiency in biofilms.

To answer this question, two factors must be considered: (i) comparison between FRAP and FCS measurements and (ii) the difference in the concentration of antibiotics each method requires (a few hundred nanomoles for FCS and 3 to 10 times more than that for FRAP). With the low concentration range used for FCS experiments, we have obtained correlation curves corresponding to the biofilm motion due to a total interaction of BODIPY-vancomycin with the biomass. In the context of FRAP experiments, as mentioned above, a significant percentage of the antibiotic (50 to 60%) can freely diffuse in all parts of the biofilm, in agreement with an excess of antibiotic relatively to the concentration fixed on biofilm components. Thus, it seems unlikely that the lack of vancomycin bioavailability could explain the failure of the antibiotic activity in such biofilms. Shielding mechanisms of biofilms that derive from the specific physiology of embedded bacteria should be further studied.

In conclusion, we have demonstrated here that advanced fluorescence imaging methods (time-lapse, FLIM, FRAP, and FCS) represent versatile and nondestructive tools for studying the diffusion-reaction of molecules as small as antibiotics through the depth of biofilms. The time-lapse approach is accessible using current commercial confocal microscopes, and its advantage lies in the possibility of monitoring the penetration of fluorescent antibiotics over time in all parts of the biofilm. The FRAP, FLIM, and FCS methods require more experience in data analysis and/or additional instrumental acquisition [START_REF] Bridier | Deciphering biofilm structure and reactivity by multiscale time-resolved fluorescence analysis[END_REF]. However, they allow diffu-sion-reaction measurements when the antimicrobials have reached equilibrium inside the biomatrix (within several hours). The use of these correlative time-resolved fluorescence microscopy methods has expanded our knowledge on the role of the biofilm structure on the local diffusion-reaction properties of antibiotics.

FIG 1

 1 FIG 1 (a) Normalized fluorescence autocorrelation curves g() for BODIPY (OE) and BODIPY-vancomycin (OE) in solution. The inset shows the correlation curve g() for BODIPY () in the presence of S. aureus planktonic cells. (b) Normalized fluorescence autocorrelation curves g() for S. aureus planktonic cells without (ᮀ) or with (o) BODIPY-vancomycin revealing interaction of vancomycin with S. aureus cell wall. (c) Fluorescence time traces for S. aureus planktonic cells without (inset) and with BODIPY-vancomycin.In the presence of labeled vancomycin, the fluorescence intensity (given in arbitrary units [a.u.]) is much higher, confirming the presence of the antibiotic in the excited volume.

FIG 2 FIG 3

 23 FIG 2 (a) Fluorescence lifetime decay for BODIPY-vancomycin in solution (solid triangles), with S. aureus planktonic cells (gray triangles), and in biofilms (open circles). (b) FLIM representation (in an x-y plane) of BODIPY-vancomycin inside S. aureus biofilms (50 by 50 m 2 ). The white scale bar represents 10 m.

FIG 4

 4 FIG 4 Time course of BODIPY-vancomycin penetration measured at the bottom of S. aureus ATCC 6538 (a) and ATCC 27217 (b) biofilms. The relative intensities correspond to a normalization of the maximum intensity value to 1. (c and d) Corresponding fluorescence intensity images taken at the bottom of the biofilms (in an x-y plane) showing BODIPY-vancomycin penetration in the whole structure of biofilms at 400 s (c) and 1,000 s (d), when the antibiotic equilibrium is reached. The images are sectioned along two vertical and orthogonal x-z and y-z planes: the substratum-biofilm interface is the outside part of the rectangle, and the biofilm-medium interface is the one close to the square fluorescence intensity image. Scale bar, 20 m.

FIG 5

 5 FIG 5 FRAP acquisitions for BODIPY (a to c) and BODIPY-vancomycin (d to f) inside S. aureus ATCC 27217 biofilms. (a and d) Kymogram representation (xt) of FRAP acquisitions (representation of fluorescence intensity along a horizontal line passing through the photobleached area as a function of time t) from t ϭ 0 to 75 s. (b and e) Sequence of fluorescence intensity images starting just before photobleaching and showing the beginning of the recovery phase. The time interval between two images is 265 ms. Scale bar, 5 m. (c and f) Typical fluorescence recovery curves recorded in three different zones of the biofilms showing a total fluorescence recovery for BODIPY and a partial fluorescence recovery for BODIPY-vancomycin. The fluorescence intensity is expressed in arbitrary units (a.u.).

FIG 6

 6 FIG 6 (a) Fluorescence time traces for S. aureus biofilm without (inset) and with BODIPY-vancomycin. In the presence of labeled vancomycin, the fluorescence intensity of biofilm cells increases, which corresponds to the motion of fluorescently cell aggregates in the excitation volume. (b) Normalized fluorescence autocorrelation curves g() for BODIPY-vancomycin interaction with S. aureus cells inside biofilm. The measurements were obtained in differ-ent zones: ϳ3 m (o), ϳ10 m (), and ϳ25 m (OE) from the biofilmcoverslip interface. The fit of the curves (straight lines) was obtained using equation 2. The gray curve corresponds to the motion of S. aureus biofilm without BODIPY-vancomycin. (Inset) g() for BODIPY diffusion inside S. aureus biofilm. (c) Fluorescence autocorrelation curves g() for BODIPY-vancomycin diffusion with P. aeruginosa biofilm. The acquisitions were performed inside (ᮀ, zone 1) and outside (OE, zone 2) the mushroom structure of the biofilm (the zones are defined in Fig.S1cin the supplemental material). The fit of the curves (straight lines) was obtained using equation 2.

TABLE 1

 1 Diffusion times, their corresponding calculated diffusion coefficients, and fluorescence lifetimes for BODIPY and BODIPY-vancomycin in water and in the presence of S. aureus planktonic cells a Diffusion times ( 1 and 2 ) obtained from fitting with the equation from the fluorescent correlation curves, their corresponding calculated diffusion coefficients (D 1 and D 2 ), and the fluorescence lifetimes for BODIPY and BODIPY-vancomycin in water and in the presence of S. aureus plankton cells were determined. The percentages given in parentheses correspond to the ratio of molecules diffusing with the indicated time. b Values are expressed as means Ϯ the SEM or as a range, where applicable. Percentages are indicated in parentheses where applicable.

		Mean Ϯ SEM or range b
		In water		With S. aureus planktonic cells
	Method	BODIPY	BODIPY-vancomycin	BODIPY	BODIPY-vancomycin
	FCS
	1 (s)	38 Ϯ 6 (100)	52 Ϯ 11 (92)	54 Ϯ 9 (87)	30,000-100,000 (100)
	D 1 (m 2 /s)	395 Ϯ 60	290 Ϯ 60	278 Ϯ 46	0.15-0.50
	2 (ms)		1.26 Ϯ 0.62 (8)	30-100 (13)
	D 2 (m 2 /s)		12 Ϯ 6	0.15-0.50
	FLIM (ns)	5.7 Ϯ 0.2	5.7 Ϯ 0.2	5.7 Ϯ 0.2	4.5 Ϯ 0.2

a

TABLE 2

 2 Diffusion coefficients of BODIPY and BODIPY-vancomycin measured in ATCC 6538 and ATCC 27217 S. aureus biofilms using different time-resolved fluorescence techniques

			Mean Ϯ SEM a
				BODIPY-vancomycin
				ATCC 6538	ATCC 27217
	Method	Variable	BODIPY	strain	strain
	Time-lapse	D p (m 2 /s)	Ͻ140	2.0 Ϯ 0.7	0.8 Ϯ 0.3
	FRAP	D (m 2 /s)	180 Ϯ 60	0.5 Ϯ 0.2	0.2 Ϯ 0.1
	FCS	D (m 2 /s)	242 Ϯ 36	Biofilm motion

a Values are indicated as means Ϯ the SEM where applicable.

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ACKNOWLEDGMENTS

S.D.O. was funded by the CNRS, France.

We thank Antoine Monsel, physician at the Pitié Salpêtrière Hospital (Paris, France), for valuable discussions. We thank Céline Merlin for English revision of the manuscript.