Chahrazed Bouhini
email: chahrazed.bouhini@univ-st-etienne.fr

Mathias Géry
email: mathias.gery@univ-st-etienne.fr

Keywords: Personalized Information Retrieval, User's Profile, Social Data, Folksonomies

teaching and research institutions in France or abroad, or from public or private research centers.

I. INTRODUCTION

Classical information retrieval systems are based on the keyword search: given a collection of resources and a user information need, they aim to provide relevant resources to the user. Most of the time, this information need is expressed with a query composed of a few keywords. Usually, less than three words, because few users are able to formulate their needs with complex queries [START_REF] Jansen | Real life information retrieval: A study of user queries on the web[END_REF], [START_REF] Kamps | Structured queries in XML retrieval[END_REF], [START_REF] Kazai | Users' perspectives on the usefulness of structure for XML information retrieval[END_REF]. However, it has been shown that the queries can be ambiguous since users with different needs can use the same query even if they expect different relevant resources [START_REF] Sanderson | Ambiguous queries: test collections need more sense[END_REF]. A solution to enhance the information retrieval process, without altering the way that the users specify their requests, is a personalized information retrieval which takes into account not only the query but also other information given, directly or not, by the user [START_REF] Agichtein | Improving web search ranking by incorporating user behavior information[END_REF]. The advent of the Web 2.0, with social tagging systems which allow the users to annotate resources, has notably provided complementary information useful to disambiguate the query and personalize the search. This lead notably to Personalized Information Retrieval (PIR) which can be defined as the incorporation of information related to social networks into the information retrieval process [START_REF] Kirsch | Social information retrieval[END_REF]. To this end, the main task of Personalized Information Retrieval is to exploit the user's profile and integrate it during the IR process. This requires to adapt the usual IR models and systems in order to deal with this user's social data.

In this article, we propose to generate the user's profile from his social annotations. More specifically, once the user's profile is generated, we investigate the way to exploit the information provided by the term distribution within the user's profile. Then, we present social IR models called BM 25S prof ile , BM 25S ScoreComb and BM 25S F reqComb , which integrate this user's profile during the querying step.

In order to evaluate our social IR models that take in consideration the users' social data, we built a social IR test collection providing a user-centered data (user-centered relevance judgments). This collection is based on the social annotations extracted from the social collaborative bookmarking system Delicious1 . This article is organized as follows: we discuss some related works in Section II and we explain the motivations of our work in Section III. In Section IV, we present our methodological framework by describing the approach that we used to generate the user's profile and we detail the main contribution of our work which consists in integrating this user's profile in the querying step. Further, we present in Section V the results of the experiments conducted on the social test collection we built, described and we conclude with perspectives in Section VI.

II. RELATED WORK

Personalized Information Retrieval (PIR) attempts to extend classical IR by taking into consideration the user's profile within his social network. This profile may be integrated in order to refine the query, like in query expansion, or during the indexation and the ranking of the documents. In this last case, considered in this article, the ranking of a document depends not only on the matching between the document and the query, like in classical IR, but also on the matching between the user's interest and the document.

A. User's profiles

The user's interests are generally summarized by his profile, which may be built from his social annotations. These annotations correspond to short descriptions (or tags) given by the user to the resources. The user's profile can also integrate the tags used by the user's neighbors in his social network. In several models, notably inspired by the Vector Space Model, the document, the query and the profile are then described by vectors defined in the same space: the terms. The matching between a query and a document as well as the matching between the user's profile and the document are computed with the cosine similarity. This approach has been proposed in different works [START_REF] Diederich | Finding communities of practice from user profiles based on folksonomies[END_REF], [START_REF] Xu | Exploring folksonomy for personalized search[END_REF], [START_REF] Cai | Personalized search by tag-based user profile and resource profile in collaborative tagging systems[END_REF], [START_REF] Vallet | Personalizing web search with folksonomy-based user and document profiles[END_REF].

B. Tags based IR

Diederich and Iofciu propose an approach where the search exploits the tags put on the document by the users [START_REF] Diederich | Finding communities of practice from user profiles based on folksonomies[END_REF], but the search is not really personalized because the same set of documents is returned to the users who gave the same query, whatever their profiles. Indeed, the ranking of the document depends on the similarity between the query and the tagbased description of the document and it is not based on the appearance of the query terms into the document like in a classical IR process.

C. Combining tags and profiles for Personalized IR

The vector space model has been adapted by Xu et al. for personalized IR [START_REF] Xu | Exploring folksonomy for personalized search[END_REF]. As explained previously, the query, the document and the user's profile are represented by vectors in a space defined by the tags appearing in the annotations. The weight of a tag in the vectors associated to the user's profile and the resource are computed with the usual TF.IDF formula. Then, two scores are computed using the cosine, on the one hand the similarity between the document and the query and, on the other hand, the similarity between the document and the user's profile. Each score is used to generate a ranked document list, the first one in function of the query and the second one in function of the user's profile. The relevance of the document is finally estimated by a global score defined as a linear combination of the previous scores where the Weighted Borda Fuse algorithm (WBF) is used to aggregate the rankings.

The personalized model of Xu et al. [START_REF] Xu | Exploring folksonomy for personalized search[END_REF] has been adapted by Vallet et al. [START_REF] Vallet | Personalizing web search with folksonomy-based user and document profiles[END_REF] and by Cai and Li [START_REF] Cai | Personalized search by tag-based user profile and resource profile in collaborative tagging systems[END_REF]. In the variant introduced by Vallet et al., the similarity between a user and a document is computed with BM25 instead of TF.IDF. The difference between the proposition of Cai and Li and the model of Xu et al. lies in the combination of the query score and of the user score: the former work proposes to compute the final score as the product of these two scores, when the latter proposes to obtain it by an aggregation of the rankings. Moreover, in the approach of Cai and Li, the vectorial representation of the resource differs slightly from the one adopted by Xu et al.. Kashyap et al., based on a linear combination of the ranking given by a classical search engine and the social ranking [START_REF] Kashyap | Sonetrank: leveraging social networks to personalize search[END_REF]. However, in this model, the social ranking is done using an adaptation of the PageRank on a graph representing the relationships between the documents, the queries and the users or groups of users. If the graph is a rich model of the interactions between the different components of a PIR, it is also more complex to handle.

Another model has been proposed by

D. Discussion

From these previous works, we retain the idea that it is important to exploit the social annotations of the user. So in our models, we propose also to combine the social data and the query data. However, we consider that the representation of the documents can not be based only on the tags like in the previously mentioned works. Moreover, we think that combining the document content with this user's profile requires IR techniques that are able to handle large textual documents. This led us to introduce an original approach presented in the following sections.

III. PERSONALIZED IR EXPLOITING FOLKSONOMIES

Our approach is based on several assumptions as detailed below.

A. Information need: query and personal interests

Almost all test collections, in IR research, assume that queries have a single interpretation representing the information need expressed by one user, which is implicitly defined in his relevance judgments [START_REF] Sanderson | Ambiguous queries: test collections need more sense[END_REF]. However, in practice this is not necessary the case. For this reason, in this paper we propose a framework for Personalized Information Retrieval based on folksonomies. Such a system should be able to handle ambiguous queries, i.e. queries having potentially several interpretations representing different information needs.

For example, suppose that two users Alice and Bob ask the same query q = "smartphone android" (cf. Table I).

We consider two documents d 1 and d 2 ; each document contains one query term, but smartphone is more important than android in the first document since d 1 contains only smartphone, and android is more important than smartphone in the second one since d 2 contains only android. Assuming that the two query terms have the same importance, a classical IR system should estimate that d 1 is equally relevant as d 2 for the query "smartphone android". However, depending on the user and his personal interests, the information need behind this query may focus either on the term smartphone or on the term android. Now, if we suppose that Alice is mainly interested in smartphone devices, then her information need is probably centered around smartphones with an opening on Android, and thus the query term smartphone should be more important than the query term android. On the other hand, if Bob is mainly interested by the Android operating system, then his information need is probably centered around Android, and thus the query term android should be more important than the query term smartphone.

A personalized information retrieval system should be able to identify the user's personal interests, in order to better interpret the information need behind his queries, and returns lists of relevant documents to the users depending on their personal interests. In our example, a Personalized Social IR system should consider d 1 as more relevant than d 2 for Alice and the opposite for Bob.

B. Folksonomies and user's profile

We assume that folksonomies may be exploited in order to build the informational social profile of the user that could represent the user's interests and that could help the system to handle ambiguous queries and return personalized results to the user. As pointed out in related literature, the user's profile can be inferred from his social annotations [START_REF] Au-Yeung | A study of user profile generation from folksonomies[END_REF], [START_REF] Szomszor | Semantic modelling of user interests based on cross-folksonomy analysis[END_REF].

C. Integrating user's profile

We think that the integration of the user's profile within the IR model is an important part of the personalization approach.

Since we exploit the social information about the user to generate his profile, we assume that the important terms representing the user's interests should appear in this profile. Thus, reweighting such important terms when they are found within the document, should improve the document relevance score and allow to return the personalized relevant documents.

Then, the aim of our approach is to exploit together several sources of information:

• The textual content of the documents, containing thousand of terms; • The explicit user's information need, expressed as a short query composed of a few terms; • The implicit user's information need, which is somewhere expressed by his profile.

D. Weighting large textual documents

One aim of this work is to handle textual documents containing thousands of terms, unlike most related works which only handle small sets of tags describing the document. For this reason, a basic tf.idf weighting function is not suitable: we need to use term weighting function that is designed to process large textual documents and the well-known Okapi BM25 weighting function seems to be one of the best choice.

The behavior of such non-binary functions, on the document side as well as on the query side, aims at giving less importance to each new occurrence of a term in the same document: there is a significant difference if a term appears two times in the same document (and then the increase of its weight should be significant), but there is almost no difference between 1000 and 1001 occurrences of a term in the same document (and then the increase of its weight should be negligible). This behavior is called "saturation" [START_REF] Robertson | The Probabilistic Relevance Framework: BM25 and Beyond[END_REF].

E. Weighting short queries and user's profile

On the query side, in classical information retrieval, the terms have usually a binary weight: the term appears in the query (term frequency = 1) or the term does not appear (term frequency = 0).

In the case of profiles, including several occurrences of the same term, it seems interesting to consider the term frequencies, like weighting functions do on the document side. Especially, it seems important to provide the saturation effect of a non-linear weighting function.

F. Combining binary queries with user's profile

Combining the user's profile (term frequencies) with a query (binary) and then computing the matching score with textual documents containing thousands of terms raises different issues than computing the matching between binary queries and textual documents (classical IR) or than only combining user's profile and the document's profile represented by a set of tags, like for instance in the work of Cai and Li [START_REF] Cai | Personalized search by tag-based user profile and resource profile in collaborative tagging systems[END_REF].

Especially, combining binary query with user's profile raises an important issue: how to equilibrate the importance of these two different kind of information?

In this work, we propose to study four different strategies:

• the first one (called classical BM 25) considers only the explicit user's information need, expressed as a short binary query composed of a few terms; • the second one (called BM 25S prof ile ) considers only the implicit user's information need, expressed by its nonbinary profile composed of many terms; • the third one (called BM 25S ScoreComb ) combines the initial binary queries and the user's profile at a scoring level; • the fourth one (called BM 25S F reqComb ) combines the initial binary queries and the user's profile at a term frequencies level. Our approach is inspired by the work of Robertson et al., who have shown, while combining several textual fields that compose a structured document, that the "term frequencies level" strategy is theoretically and experimentally better than the "scoring level" strategy [START_REF] Robertson | Simple BM25 extension to multiple weighted fields[END_REF]. One aim of this work is to check this conclusion in the context of PIR.

IV. PERSONALIZED INFORMATION RETRIEVAL MODELS

We present the well known Information Retrieval model BM25 (cf. Section IV-B), on which are based our Personalized Information Retrieval models, as well as our three PIR models that exploit the user's profile in order to refine the user's query:

• BM 25S prof ile (d, u) (PIR model): this model returns a ranked list of documents that are relevant for a given user u considering only his profile. Thus, the binary query q is simply replaced by u in equation ( 6).

• BM 25S ScoreComb (d, q, u) (combined PIR model): this model returns a ranked list of documents that are relevant for a given user u considering his binary query q combined at the scoring level with his profile. • BM 25S F reqComb (d, q, u) (combined PIR model): this model returns a ranked list of documents that are relevant for a given user u considering his binary query q combined at the term frequencies level with his profile, as recommended by Robertson et al. [START_REF] Robertson | Simple BM25 extension to multiple weighted fields[END_REF].

A. Notations

We represent the "social tagging data", also known as Folksonomies [START_REF] Vallet | Personalizing web search with folksonomy-based user and document profiles[END_REF], by a tuple R S = < U, T, D, A, Rel >, where:

• U is a set of social network users; • T is a set of index terms; • D is a set of documents on the Web (images, videos, Web pages, etc.); • A is a set of social annotations;

• and Rel ⊆ U × U is a set of relationships between pairs of users.

Rel is such that (u, u y ) ∈ Rel if there is a social relationship between the users u and u y . The users related to u are typically those declared explicitly by u as his neighbors where neighborhood(u) = {u y / (u, u y ) ∈ Rel}.

We note also that:

• a document d ∈ D is represented by a set of terms t ∈ T and a term t may appear one or several times in d; • tf d,t is the term frequency of t d; • w d,t is the weight of a term t for a document d, computed using its term frequency tf d,t ; • a z ∈ A with a z = < d, u, T z > is the annotation of the user u for the document d using a subset of terms T z ⊂ T .

On the query side, we note that:

• Q is a set of users' queries;

• each query q ∈ Q is represented by a set of terms t ∈ T ;

• Qrels is a set of global relevance judgments (i.e. independently of the user); • qrels q ⊂ D denotes the set of relevant documents for q independently of the user; • Q U C is a set of couples (q, u) where the query q is issued by the user u to express his personal information needs; • Qrels U C is a set of user-centered relevance judgments (i.e. dependently to a given user); • qrels q,u ⊂ D is the set of relevant documents for the query q and the user u.

B. BM 25: classical Information Retrieval weighting function

We choose to build our Personalized Information Retrieval models on the IR weighting function BM25 [START_REF] Robertson | Okapi at trec'4[END_REF], which is one of the most used indexation models in the IR research benchmarks such as INEX2 , TREC3 , etc. This IR weighting function is composed by three parts:

• Aboutness: T F d,t estimates the aboutness of the document d considering the term t (cf. equation 1). • Discrimination: DF t estimates the discriminating power of the term t to the documents (cf. equation 2). • Query terms: QT F q,t estimates the importance of the term t within the query q (cf. equation 3).

Aboutness (T F d,t ), term discriminating power (DF t ) and query terms importance (QT F q,t ) are defined as follows [START_REF] Robertson | Okapi at trec'4[END_REF]:

T F d,t = (k 1 + 1) × tf d,t k 1 × (b -1) + b × dl avgdl + tf d,t (1)
DF t = log N -df t + 0.5 df t + 0.5 (2)
QT F q,t = (k 3 + 1) × tf q,t k 3 + tf q,t (3)
where:

• dl is the document length of d and avgdl is the average documents length. • tf d,t is the term frequency of t within the document d.

• k 1 is the saturation parameter of tf d,t .

• b is the length normalization factor.

• N is the total number of documents in the corpus.

• df t is the number of documents containing the term t.

• tf q,t is the term frequency of t within the query q.

• k 3 is the saturation parameter of tf q,t .

The weight w d,t of a term t within a document d is computed by the BM 25 weighting function as shown in equation 5:

w d,t = T F d,t × DF t (4) BM 25(d, q) = t∈q∩d w d,t × QT F q,t (5)
The whole BM25 scoring formula is shown in equation 6. We assume that the initial query terms are not weighted, i.e. ∀q ∈ Q, ∀t ∈ q, tf q,t = 1. Thus, in the PIR models which do not consider the user's profile and exploit only the initial query, QT F q,t can be ignored. In this case, the global IR score of a document for a query is given by equation 5 with QT F q,t = 1.

C. User's profile

The user's profile may contain different information types (annotations, comments, citations, social relationships, etc.). In this work, we generate the user's profile from the terms in his annotations. The user's profile prof ile(u) is the set of terms which occur within his social annotations:

BM 25(d, q) = t∈q∩d (k 1 + 1) × tf d,t k 1 × b -1 + b × dl avgdl + tf d,t × log N -df t + 0.5 df t + 0.5 × (k 3 + 1) × tf q,t k 3 + tf q,t (6)
prof ile(u) = {t ∈ T z / a z = < d, u, T z > ∈ A u } (7)
where A u is the set of social annotations of u.

The user's profile may contain several occurrences of the same term. Thus we can compute the term frequency tf u,t for a given term t that has been used by u to annotate the documents.

D. BM 25S prof ile (d, u): Personalized Social IR by exploiting only the user's profile

Our aim is to take into account the user's profile during the querying step, in order to personalize the IR process. In BM 25S prof ile (d, u), we replace the query by the user's profile in the scoring function BM 25(d, q), in order to retrieve a ranked list of documents that are relevant for a given user u considering only his profile. The query terms are replaced by the user's profile terms: in other words tf q,t is replaced by tf u,t in equation 6.

Unlike the queries, which are composed generally of a few terms, the user's profile contains tens, hundreds or sometimes hundreds of terms. We make the assumption that this implicit user's information need, which is more complex, has to be represented by a vector of term weights, instead of binary weights. We think that a suitable weighting function should distinguish the most important terms from the user's profile. We think also that it should include a saturation effect of the term frequencies, in the same way that the saturation effect of the IR weighting formula BM25 (parameters k 1 and k 3 ). Then, we propose three versions of the scoring function BM 25S prof ile (d, u). These scoring functions are computed using the BM25 function (cf. equation 6):

• In the first model BM 25S prof ile-bin (d, u) (binary profile), k 3 = 0 in equation 6 corresponds to a maximum level of saturation: the user's profile is represented by a binary vector; • In the second model BM 25S prof ile-tf (d, u) (term frequencies profile), k 3 = 1000 in equation 6 corresponds to set off the saturation level on tf u,t [START_REF] Jones | A probabilistic model of information retrieval: development and comparative experiments, part 2[END_REF]: the user's profile is represented by a vector of term frequencies; • BM 25S prof ile-w (d, u) (weighted profile), k 3 tuned in equation 6 corresponds to a moderate saturation of tf u,t in order to reach the optimal saturation level: the user's profile is represented by a vector of term weights.

This way, k 3 plays the same role on the user's profile weighting than k 1 on the documents weighting. It remains two differences between terms weighting in the documents and in the user's profile: the first one is that there is no DF component in the user's profile weighting. However, as this component still appear at the document level, it is not useful to use it twice. The second one is that there is no document length normalization in the user's profile weighting, assuming that the users' profiles lengths are more homogeneous than the documents ones.

E. BM 25S ScoreComb (d, q, u): Personalized Social IR by combining query and profile at the scoring level This first combined PIR model, BM 25S ScoreComb , combines query and user's profile at the scoring level. Given a query q and a user u, for each document d the global score BM 25S ScoreComb (d, q, u) is composed of two parts:

• in the first part, on the query side, the classical relevance score BM 25(d, q) is computed between the query and a document as detailed in section IV-B; • in the second part, considering the user's profile side, a PIR relevance score BM 25S prof ile (d, u) is computed between the (non-binary) user's profile and a document as detailed in section IV-D. This PIR score can be computed with one of our three PIR scoring functions BM 25S prof ile-bin (d, u), BM 25S prof ile-tf (d, u) or BM 25S prof ile-w (d, u). Then, a combined PIR relevance score BM 25S ScoreComb (d, q, u) is computed as shown in the equation 8, based on a simple linear combination with the query relevance score and the profile relevance score.

BM 25S ScoreComb-X (d, q, u) = BM 25(d, q) +α 1 × BM 25S prof ile-X (d, u) (8)
where X is "bin", "tf " or "w", referring to one of the three variants of BM 25S prof ile (d, u): BM 25S prof ile-bin (d, u), BM 25S prof ile-tf (d, u) or BM 25S prof ile-w (d, u).

F. BM 25S F reqComb (d, q, u): Personalized Social IR by combining query and profile at the term frequencies level

This second combined PIR model, BM 25S F reqComb (d, q, u), combines query and user's profile at a terms frequencies level. The user's profile term frequencies are combined with the query term frequencies by a simple linear combination. This leads to replace QT F q,t in equation 4 by the QT F q,u,t defined in equation 9.

QT F q,u,t = (k 3 + 1) × [tf q,t + α 2 tf u,t ] k 3 + (tf q,t + α 2 tf u,t ) (9)
And the PIR relevance score BM 25S F reqComb (d, q, u) of the document d for the query q and the user u is computed with the weighted variant of the BM25 weighting function, as follow: [START_REF] Vallet | Personalizing web search with folksonomy-based user and document profiles[END_REF] Like in our previous PIR model, we propose three variants of our combined PIR model: BM 25S F reqComb-bin with k 3 = 0, BM 25S F reqComb-tf with k 3 = 1000 and BM 25S F reqComb-w with k 3 tuned.

BM 25S F reqComb (d, q, u) = t∈q∩d w d,t × QT F q,u,t

V. EXPERIMENTS AND RESULTS

Our aim in conducting these experiments is to evaluate, on a Personalized Information Retrieval test collection, PIR models which take in consideration the user's profile. An IR test collection is composed by a set of documents, a set of queries and a set of relevance judgments (i.e. the list of relevant documents for each query). A PIR test collection needs also user-centered data: a set of users asking the queries and a set of user-centered relevance judgments, i.e. the list of relevant documents for each couple (user, query).

A. Personalized IR test collection

With the lack of a PIR test collection providing a list of relevant documents for each query and for each user, we have built a test collection DelP IR based on Web documents and users' annotations extracted from the social collaborative bookmarking network Delicious.

We have collected 161,059 documents in 207,304 bookmarks. In Delicious, the documents are annotated by the users. So we have been able to collect the 68,600 terms associated to these 161,059 documents by 8,069 users of Delicious. We have created automatically 100 queries: each query is composed of 3 terms occurring frequently together in the annotations collected from Delicious. In fact, the 100 triples of terms having the highest Jaccard Index. We have chosen three terms since in practice, most of the time, users formulate their needs with few words [START_REF] Kamps | Structured queries in XML retrieval[END_REF]. These 100 queries, called "global queries" Q, are issued by 1,118 users, so we obtained a set Q U C of 15,010 couples (query, user).

Then we have generated Qrels, the set of 107,934 global relevance judgments, i.e. 107,934 couples (q, d) where the document d is relevant considering the query q, independently of the users. A document is globally relevant if it has been annotated by any user with at least 2/3 of the query terms in the same annotation, since three terms seems too restrictive.

And finally, we have generated Qrels U C , the set of 137,381 user-centered relevance judgments, i.e. 137,381 triples (q, d, u) where the document d is relevant considering the query q, for the user u. A document is user-relevant if it has been annotated by the user with at least 2/3 of the query terms in the same annotation. Some statistics about the generated PIR test collection are summarized in the Table II.

B. Filtering

We have applied two kind of filters to the PIR test collection:

1) Documents filter (F Doc ): we have kept only the couples (query, user) with at least 10 relevant documents (|qrels q,u | ≥ 10) so that we have obtained 6,361 couples (q, u) in the set Q U C . This led to a reduction of the number of users (710 users left) and thus a reduction of the number of user-centered relevance judgments (93,520). 2) "Hard query" filter (F HQ ): we have observed that a lot of the generated queries seem to be very "hard queries", at least for a classical BM25 based IR system. Thus, in order to obtain queries similar to the queries of real users, we have eliminated the most "hard" ones. Using a classical well-tuned BM25 based IR system, we have kept the couples (query, user) obtaining an AP (average precision value) above 0.1%. We have obtained 1,363 couples (q, u) in the set Q U C . This led to a reduction of the number of users (443 users left) and thus a reduction of the number of user-centered relevance judgments (31,910).

Some statistics about the first step of filtering F Doc , and about the final PIR test collection DelP IR obtained after the two steps of filtering F Doc and F HQ , are summarized in the Table III.

The results shown in this section have been obtained by evaluating the classical model and the PIR models on the filtered PIR test collection data DelP IR using F Doc and F HQ . This filtered PIR test collection is composed of 161,059 documents, 443 users, 98 global queries with 107,923 global relevance judgments and 1,363 user-centered queries with 31,910 user-centered relevance judgments. We have evaluated the rankings produced by our PIR models with only usercentered data (Q U C and Qrels U C ), since the PIR model is not suited to handle global queries.

C. Evaluation measures

We have used the traditional evaluation measures "Precision at 10% of recall" (P [0.1]) and "Mean Average Precision" (M AP ) which are the most used evaluation measures for evaluating the classical IR systems [START_REF] Manning | Introduction to Information Retrieval[END_REF].

D. Evaluation results: BM 25

The evaluation results of the classical BM 25 model are shown in Table IV.

In all our experiments we have tuned the parameters of the models (i.e. b and k parameters of BM25) using a grid-based 2D optimization, as proposed by Zaragoza et al., [START_REF] Zaragoza | Microsoft cambridge at TREC 13: Web and hard tracks[END_REF]. This well-tuned BM 25 is a strong baseline for our PIR evaluation described in the next sections. These results show that the PIR model BM 25S prof ile (d, u) that replaces the user's initial query by his profile, provides better results than the baseline BM 25(d, q) when the saturation level is balanced (BM 25S prof ile-w (d, u)), considering the MAP evaluation measure: 0.0116 vs 0.0108. The evaluation results of the variants BM 25S prof ile-bin , having a maximum saturation level (k 3 = 0), and BM 25S prof ile-tf , with no saturation, are worse than the baseline.

Considering the precision at 10% of recall (P[0.1]), the PIR model provides less good results than the baseline.

F. Evaluation results: BM 25S ScoreComb

The evaluation results of our PIR model BM 25S ScoreComb with its variants BM 25S ScoreComb-bin , BM 25S ScoreComb-tf and BM 25S ScoreComb-w compared to the baseline BM 25 are shown in Table VIII.

The results of our first combined PIR model BM 25S ScoreComb which combines the classical IR score BM 25(d, q) and the social score BM 25S prof ile (d, u), that considers only the matching between the document and the user's profile, are better (M AP = 0.0140 and P [0.1] = 0.0308) than the results provided by using only the classical model (baseline: BM 25(d, q)) or only a social score (BM 25S prof ile (d, u)).

We observe that, unlike in BM 25S prof ile-bin vs BM 25S prof ile-tf and BM 25S prof ile-w , the results of the binary variant BM 25S ScoreComb-bin are better than the results of the other two variants (BM 25S ScoreComb-tf and BM 25S ScoreComb-w ).

G. Evaluation results: BM 25S F reqComb

In this section, we present the evaluation results of the PIR model BM 25S F reqComb which exploits the user's profile by combining, within the query sent to the PIR system, the initial query terms and the user's profile terms.

The results, shown in Table IX, obtained with BM 25S F reqComb , are better than the baseline (BM 25(d, q): M AP = 0.0108 and P [0.1] = 0.0253) with all its variant (BM 25S F reqComb-bin , BM 25S F reqComb-tf and BM 25S F reqComb-w ).

We notice that the binary combination BM 25S F reqComb-bin having a maximum level of saturation of terms within the query, gives better results (M AP = 0.0132 and P [0.1] = 0.0292) comparing to the other variants BM 25S F reqComb-tf with no saturation and 0.0108 0.0253 BM 25S F reqComb-bin (d, q, u) 0.0132 0.0292 α 2 = 0.07 BM 25S F reqComb-tf (d, q, u) 0.0119 0.0258 α 2 = 0.004 BM 25S F reqComb-w (d, q, u) 0.0128 0.0287 α 2 = 0.004 BM 25S F reqComb-w having a balanced saturation level (k 3 = 0.07).

H. Discussion

The results detailed in Table VI • PIR model which takes into account the user's profile, enhances the relevance score results using the MAP measure which is considered as a global evaluation metric. • Combining the scores BM 25(d, q) and BM 25S prof ile improve the evaluation results comparing to using each score apart. Moreover, we notice that the PIR model BM 25S ScoreComb that combines at the scoring level, the query and the user's informational social profile, gives better results than the PIR model BM 25S F reqComb combining the query and the user's profile at the frequencies level:

• BM 25S F reqComb : M AP = 0.0132, P [0.1] = 0.0292; • BM 25S ScoreComb : M AP = 0.0140, P [0.1] = 0.0308. This result does not confirm the conclusion of Robertson et al., according which the "term frequencies level" strategy is better than the "scoring level" strategy [START_REF] Robertson | Simple BM25 extension to multiple weighted fields[END_REF].

VI. CONCLUSION

We have presented an approach to build the user's profile from his annotations using a dataset collected from the social bookmarking network Delicious. We proposed different ways of considering the occurrences of the important terms for the user within his profile: binary, term frequencies and weighted profile, each corresponding to a different level of saturation. We have investigated then, the different ways of integrating the user's profile.

• Firstly, we have presented a simple PIR model BM 25S prof ile that replaces the initial query terms by the user's profile terms. • Then, we proposed to combine different scores: the thematic score (classical BM 25S score of a document for a query) and the social score BM 25S prof ile within a PIR model we called BM 25S ScoreComb . • Afterward, we attempted to combine the initial query terms and the user's profile at the frequencies level, by extending the initial query in our PIR model called BM 25S F reqComb .

We evaluated our Personalized Information Retrieval models on a PIR test collection we have built using the annotations extracted from Delicious. Our preliminary results showed that the PIR models improve the user-centered search (i.e.: for a given query, the list of relevant documents depends on the user) by providing better scores than the baseline.

The PIR model that considers the user's profile, allows to better find the relevant documents for the user than the IR model which does not consider the user's profile.

As future works, we plan to extend the user's profile with further social data including the neighborhood's annotations and experiment different combinations and weightings of the user's profile terms. It could be also interesting to explore the impact of more complex queries composed of more than three terms or based on concepts instead of terms.

TABLE III PIR

 III TEST COLLECTION DATA DelP IR USING DIFFERENT FILTERS.

			DelP IR
		F Doc filtering F Doc and F HQ filtering
	|D|	161,059	161,059
	|U |	710	443
	|Q|	98	98
	|Qrels|	107,923	107,923
	|Q U C |	6,361	1,363
	|Qrels U C |	93,520	31,910

TABLE IV BM

 IV 25 EVALUATION RESULTS.

		M AP		P [0.1]
	BM 25(d, q)	0.0108		0.0253
		TABLE V
	TUNED BM25 PARAMETERS VALUES.
		M AP		P [0.1]
		b	k	b	k
	BM 25(d, q)	0.6	12	0.6	30
		TABLE VI
	BM 25S prof ile EVALUATION RESULTS.
			M AP	P [0.1]
	BM 25(d, q)		0.0108	0.0253
	BM 25S prof ile-bin (d, u) 0.0102	0.0213
	with k 3 = 0
	BM 25S prof ile-tf (d, u)	0.0093	0.0168
	with k 3 = 1, 000
	BM 25S prof ile-w (d, u)	0.0116	0.0225
	with tuned k 3

Table V

 V shows the best values of tuned parameters b and k.E. Evaluation results: BM 25S prof ileThe evaluation results of our PIR model BM 25S prof ile with its variants BM 25S prof ile-bin , BM 25S prof ile-tf and BM 25S prof ile-w compared to the baseline BM 25 are shown in TableVI.TableVIIshows the best values of tuned parameters b and k.

TABLE VIII BM

 VIII 25S ScoreComb EVALUATION RESULTS.

		M AP	P [0.1]
	BM 25(d, q)	0.0108	0.0253
	BM 25S ScoreComb-bin (d, q, u)	0.0140	0.0308
		α 1 = 0.13	α 1 = 0.13
	BM 25S ScoreComb-tf (d, q, u)	0.0123	0.0291
		α 1 = 0.3	α 1 = 0.58
	BM 25S ScoreComb-w (d, q, u)	0.0137	0.0306
		α 1 = 0.26	α 1 = 0.46

TABLE IX BM

 IX 25S F reqComb EVALUATION RESULTS.

	M AP	P [0.1]
	BM 25(d, q)

  and Table VIII highlight two basic statements:

https://delicious.com/

INitiative for the Evaluation of XML-Retrieval (https://inex.mmci.unisaarland.de/)

[START_REF] Kazai | Users' perspectives on the usefulness of structure for XML information retrieval[END_REF] TExt Retrieval Conference (http://trec.nist.gov/)

VII. ACKNOWLEDGEMENT

We would like to acknowledge the financial support of Science Foundation Ireland (SFI) under Grant Number SFI/12/RC/2289.

Personalized Information Retrieval Models

Integrating the User's Profile Chahrazed Bouhini * † , Mathias Géry * , Christine Largeron *


Ashwini Chauhan

Jean-Marc Ghigo

Christophe Beloin
email: christophe.beloin@pasteur.fr

Study of in vivo catheter biofilm infections using pediatric central 1 venous catheter implanted in rat 2

Keywords: biofilm, catheter-related infection, rat model, bioluminescence 22 23

8

Venous access catheters used in clinics are prone to biofilm contamination, contributing 9 to chronic and nosocomial infections. So far, biofilm physiology was mostly studied in 10 vitro, due to a relative lack of clinically relevant in vivo models. Here, we provide a 11 relevant protocol of totally implantable venous access port (TIVAP) implanted in rats.

12

This model recapitulates all phenomena observed in clinic and allows studying bacterial 13 biofilm development and physiology. After TIVAP implantation and inoculation with 14 luminescent pathogens, in vivo biofilm formation can be monitored in situ and biofilm 15 biomass can be recovered from contaminated TIVAP and organs. We used this protocol 16 to study host responses to biofilm-infection, to evaluate preventive and curative anti-

17 biofilm strategies, and to study fundamental biofilm properties. For this procedure, one 18 should expect ~3h00 of hands-on time including the implantation in one rat followed by 19 in situ luminescence monitoring and bacterial load estimation.

20 21

viable bacteria ranging from 7.6 to 8.3-log CFU/mL (port) and 7.5 to 9.2-logs CFU/mL 28 (catheter) were obtained in the TIVAP. The bacterial load in the organs (lungs, heart, 29 spleen, kidneys, liver or blood) can also be determined by plating tissue homogenates.

30

To complement microbiological methods assessing biofilm pathogenesis in the rat 31 TIVAP model, electron microscopy was used in order to visualize biofilm structures 32 growing on the lumen of the catheter and on the silicone septum of the TIVAP.

33

Furthermore, we also optimized cyclophosphamide-based immunosuppression to study 34 the consequences of TIVAP-associated biofilm infection in immunocompromised hosts.

36

Relevance and applications of the in vivo TIVAP biofilm model 1 2

We showed that our TIVAP rat model recapitulates all phenomena observed in clinical 3 TIVAP-associated biofilm infections and can also be used to study the fundamental 4 mechanisms of in vivo biofilm formation.

34

ii/ catheter treatment with anti-adhesive procedure 26 . We modified the surface of 35 commercial TIVAPs composed of silicone and titanium, using methyl cellulose 36 (MeCe) as well as polyethylene glycol (PEG), two macromolecules with 1 inoculated with bioluminescent bacteria, we showed that an anti-adhesive 2 approach could constitute an efficient prophylactic strategy to control infections in 3 medical devices; 4 iii/ anti-biofilm strategies against biofilm-related infection such as vaccination. 5 6. Evaluation of curative strategies against difficult-to-treat pathogens (P. 6 aeruginosa, methicillin resistant S. aureus, possibly C. albicans), for which the 7 current clinical recommendation in case of device-related infection is costly and 8 traumatic removal of the device. These curative strategies include: 9 i/ curative catheter lock solutions active on already developed infectious biofilms 10 27,28,30 . We used EDTA as adjuvant in combination with gentamicin as a lock 11 solution to eradicate all the bacterial biofilms tested using our rat TIVAP model 28 12 and L-arginine, a basic amino-acid, combined with gentamicin to eradicate S.

13

aureus and E. coli in vivo biofilms 30 ; 14 ii/ novel anti-biofilm molecules; 15 iii/ alternative strategies (ultrasonic waves, photodynamic or phage therapies). 16 7. Evaluation of anti-thrombotic treatments since thrombosis is observed using this 17 model 26 . Previous studies showed that biofilm colonized implanted catheters are 18 at higher risk of causing thrombosis 35 . We used our model to demonstrate the need to be worn. Only an approved anesthetic respirator system approved by the ethical 7 committee should be used. Also make sure to work in well-ventilated area when using

23

(TSB) supplemented with 0.2% glucose at 37°C. Other relevant bacteria may also be 24 studied within this model. While bioluminescence is an efficient tool to follow in situ 25 colonization of the device and potential dissemination, it is also possible to estimate end-26 point bacterial load using non-bioluminescent microbial variant.

28

Optimization of inoculum size and volume

29

The inoculum dose was optimized to 10 4 cells for E. coli, 10 6 cells for P. aeruginosa and 30 S. aureus. For S. epidermidis the maximum dose that could be used was 10 8 cells but 31 did not lead to bioluminescent-detectable colonization. Currently no other bioluminescent 32 strain of S. epidermidis is available 24 . The volume of the inoculum was optimized to The described methods were successfully used in our model to test the antibiofilm 2 activity of cefazolin, gentamicin, EDTA, ethanol (70%) and L-arginine, injected alone or 3 in certain combination in catheter lock solutions 27,28,30 . Moreover, our model was 4 successfully used to evaluate the anti-adhesion characteristics of biomimetic glycocalyx-5 like polymer such as methylcellulose 26 . Empirically, the effect of any relevant 6 antimicrobial agents or anti-adhesive molecules can be evaluated using our model. In 7 vitro concentration of the antimicrobial agents and procedures to graft anti-adhesive 8 molecules on silicone and/or titanium need to be verified before in vivo evaluation in rat 9 TIVAP model. Step 1: Pre-surgical Procedures

4

1A) Day -6, reception of animals: The CD/SD (IGS:Crl) male rats (CharlesRiver) weigh 15

Critical step: if given directly with the drug, most of the rats do not eat the pain-killer and 16 can lead to health issues post-surgery. Medigel with ibuprofen can be prepared on day -17

2 and stored at 4°C but brought to room temperature before feeding the rats.

19

Step 2: Surgical Procedure (TIVAP implantation, day 0) (TIMING: ~ 55min)

20

2A) Anesthesia (TIMING: 2 minutes per rat, 5-6 minutes for a rat to sleep)

21

Rats are anesthetized using a 500µL cocktail (per 300grams of rat weight) of ketamine,

22

xylazine and acepromazine mix (7:2:1, v/v) injected intraperitoneally. 4

Since it takes ~15 minutes for a rat to be ready for surgery, meanwhile, arrange and 5 prepare the surgery area. For this,

6

2D.1) Clean the surgical hood with 70% ethanol and spread a sterile champ/sheet.

12

TIVAP kit) and 1mL syringe filled with sterile 300µL heparin.

13

2D.3) Turn on 37°C thermo-cage system for storing the rat after surgery.

14

2D.4) Turn on instrument sterilizer.

15

Critical step: keep everything you need for the surgery handy as the rats may start to 16 wake up in case of delay in the process of surgery and may be stressed.

22

2E.11: Cut the catheter to a length of 4.5 cm (catheter has markings 1cm apart) at

23

an angle not more than 30°. This is the length of catheter to be inserted in the 24 external jugular vein to reach the top of right atrium.

25

Critical step: Cutting the catheter at a higher angle will allow easy insertion of 26 catheter in the jugular vein but it will lose patency quickly due to suction of vein 27 against the bigger tilted cut while pulling the blood with syringe.

28

2E.12: Make sure that port and catheter are filled with 1X PBS. For this, fill the

29

TIVAP by slowly pushing the piston of the syringe with Huber needle inserted in to 30 port.

31

Critical step: It is important to make sure before inserting the catheter in jugular 32 vein that there is no air in the TIVAP as it can kill the rat within few minutes.

33

2E.13: While gently holding the vein with a fine forceps make a partial cut in the 34 external jugular carefully using a micro spring scissors.

Critical step: There may be lots of blood flow from the vein. In this case take wet

27

Step 3: Postsurgical care: Day +1 to Day +4 28 3A) Feeding Analgesic: New cup of Medigel + ibuprofen is kept in the cage of the rats on 29 day +1 after surgery.

30

3B) "Flush and draw": (TIMING 4 minutes anesthesia, 5 minutes flush and draw per rat).

31

3B.1) To maintain the patency of the catheters, the TIVAP is flushed every day until 32 the day of the bacterial challenge. The rat is transferred to the isoflurane box until the 33 induction of anesthesia.

34

3B.2) Meanwhile, place a sterile sheet in the laminar air flow and place the nose 35 piece connected to the isoflurane system on it.

3B.3) Place the nose of the anesthetized rat in the nose piece such that the tail is 1 away from you.

2

3B.4) Clean the skin on and surrounding the port with betadine solution and 70%

3

ethanol and allow to air dry for 4-5 minutes.

4

3B.5) Gently insert the Huber needle connected to a 2mL syringe filled with 1X PBS 5 into the port by puncturing silicon septum of the port.

6

3B.6) Slowly turn the rat on its back keeping the needle inside the port.

7

3B.7) Carefully inject about 100µL 1X PBS and then check blood reflux by slowly 8

pulling the piston of the syringe. Flush TIVAP with 1.0 to 1.5mL 1X PBS, followed by 9 locking the TIVAP with 250µL heparin (250IU/mL).

10

3B.8) Clean the skin with betadine solution and put the rat back in the cage.

11

Critical step: If the rat is not anesthetized completely, the movement of neck may 12 block the blood reflux. If isoflurane system is not available chemical anesthesia can 13 be given to rats. In this case 250µL anesthesia per 500 grams rat is sufficient. another syringe containing ~250µL heparin (500IU/ml) and lock the TIVAP.

8

Step

32

5C.1) Tail vein: (TIMING: 10 minutes per rat).

33

Anesthetize the rat as described above. In case using chemical anesthesia, inject 34 only 200µL intraperitoneally as the procedure takes only 10 minutes for injection.

35

Clean the tail with 70% ethanol and rub the tail with a sterile gauze pad to remove the 36 dead scales. Approximately 5cm above the tail end insert a tuberculin needle and slowly pull the blood. Collect the appropriate volume of blood and transfer to a 1 collection tube containing anti-coagulant (for viable bacteria count, estimation of 2 immune cell population) or tube without anti-coagulant (for cytokine analysis).

3

5C.2) Retro orbital plexus: (TIMING: 10 minutes per rat)

4

Anesthetize the rat as described above. Put the rat on comfortable table. Use a 11

Critical Step: Of the circulating blood volume, approximately 10% of the total volume can 12 be safely removed every 2 to 4 weeks, 7.5% every 7 days, and 1% every 24 hours 39,40 .

13

CAUTION: Technical expertise is required before performing blood sampling. Regarding 14 the presence of retro orbital plexus instead of sinus in rats, it is not a preferred method 15 and a minimum of 10 days must be allowed for tissue repair before repeat sampling from 16 same orbit 41 .

18

Step 6: Euthanasia and sampling: (TIMING ~45 minutes per rat)

19

After the last day of observation by bioluminescence:

20

6A) Euthanasia: (TIMING ~5 minutes per rat)

21

Euthanize the animals using a procedure validated by the ethics committee of your 22 institution (CO 2 or chemical euthanasia using IP injection of Dolethal (2mL/rat, 23 200mg/mL)). Put the unconscious rat on a sterile sheet in the laminar air flow system.

24

Clean the body of the rat with 70% ethanol. 15

All the procedure is done under the laminar air flow (P2 lab).

16

6D.1) After bioluminescence imaging, wash organs in 4mL 1X PBS buffer (in a 50mL 17 conical tube) before transferring to gentleMACS™ M tubes containing 5mL 1X PBS.

18

6D.2) Weigh all the organs for analyzing parameters per gram of organ.

19

6D.3) Homogenize the organs using gentleMACS™ M tubes with the gentleMACS™ 20

Octo Dissociator (Miltenyi Biotec). Alternatively, homogenization of each organ can 21 be done using a T25 digital ULTRA-TURRAX ® homogenizer (Ika).

22

6D.4) Homogenized organs are passed through cell strainer (70µm nylon, BD

23

Falcon).

24

6D.5) The bacterial suspension is then diluted serially, plated on agar plates and 25 incubated at 37°C for colony counts.

31

6E.

2) The catheter is cut into small pieces and a slit is made horizontally to expose 32 the lumen and transferred to a microtube containing 1 mL 1X PBS.

33

6E.

3) The septum is removed from the port using a sterile scalpel and forceps, cut 34 into small pieces and transferred to a separate tube containing 1 mL 1X PBS.

1
or put in contact with skin or eyes. It is described as a possible carcinogen and teratogen.

2

Using the chemical hood and gloves is highly recommended when using this compound. 11

Antibiotics and their concentration for lock therapy studies were chosen based on 12 recommendations made by IDSA guidelines 4 (Table 1). Efficacy of cefazolin (5,000mg/mL in 13 5000IU/mL heparin) and gentamicin lock therapies (1.0mg/mL, or 5mg/mL), EDTA

14

(30mg/mL), ethanol (70%), L-arginine (0.4%) alone or in combination is evaluated against 1

15 bioluminescent E. coli

9

(IGS: Crl) rats. Surgery was performed under laminar air flow using a surgical hood and 10 aseptic conditions were maintained throughout the surgical procedure. For ease, labels

5 6

 5 TIVAP applications from a clinical point of view: 7 1. Evaluation of current procedures used to handle TIVAP in clinical settings, 8 including skin disinfection procedures, TIVAP patency maintenance 26 , etc.

9 2 .

 2 Study of the different routes of TIVAP colonization in addition to classical endo-10 luminal colonization including: 11 i/ extra-luminal colonization of the catheter; 12 ii/ extra-luminal colonization of the port leading to subcutaneous port pocket 13 infection, a clinical situation where repeated intradermal needle punctures during 14 access to TIVAP may lead to subcutaneous bacterial port pocket infection 31 15 (20% of TIVAP-implanted rats inoculated with P. aeruginosa or S. aureus 16 developed subcutaneous infection around port 27 ); 17 iii/ catheter tip colonization via hematogenous route of infection, a situation 18 observed when TIVAP implanted patients suffer from bloodstream infection: 19 TIVAP implanted rats injected with S. aureus in the tail vein showed colonization 20 of TIVAP catheter tip 27 . Furthermore, we observed, in our TIVAP rat model, 21 chronic colonization of implanted devices with occasional catheter-related 22 bloodstream infections as detected in CVC bearing patients 27 . Thus, our rat 23 TIVAP model successfully reproduced clinical situations such as biofilm-related 24 bloodstream infections, organs colonization and port-pocket infections. 25 3. Investigation of controlled chronic infection and recurrence of infection after 26 conservative treatment.

27 4 .

 4 Development of early biofilm detection methods ("biofilm biomarkers"). Such 28 biomarkers are currently lacking and their identification could strongly facilitate 29 medical decisions.

30 5 .

 5 Evaluation of prophylactic strategies including: 31 i/ novel catheter lock solutions. The catheter lock therapy corresponds to the use 32 of highly concentrated antimicrobial (often antibiotic) solutions that dwell inside 33 the catheter for at least 12 to 24 hours 32 ;

19

  better patency in the TIVAP coated with anti-adhesive coatings compared to 20 control untreated TIVAPs 26 . 21 22 TIVAP applications from a fundamental point of view: 23 1. Study of biofilm physiology in vivo and in a clinically relevant model amenable to 24 transcriptomic, proteomic or metabolomic analyses. Biofilm-specific properties 25 such as biofilm-associated production of molecules 29 or biofilm-specific increased 26 recalcitrance to antimicrobial agents and to the immune system can be studied 27 using this model.

28 2 .

 2 Evaluation of the in vivo role of biofilm-promoting factors identified in vitro.

29 3 .

 3 In vivo study of clinically relevant biofilm-forming microorganisms other than 30 bacteria, in particular C. albicans, one of the main causes of catheter-related 31 infection.

32 4 .

 4 Investigation of the impact of the implanted host immune system on the 33 development of in vivo biofilms 27 and, reciprocally, study of the impact of biofilm 34 development on the immune system.

4

  The procedure described is designed to study bacterial biofilms using a central venous 5 catheter called TIVAP inserted in rat jugular vein. The study can be broadly divided into 6 6 stages (see Flow chart of the different steps is presented in Figure 1): One animal 7 study takes between 2-3 weeks but may be longer depending on the question to be 8 addressed, for example immunosuppression of animals will add another 4 days to a 9 study. A trained engineer, PhD student, post-doctoral fellow or researcher can perform 10 all stages of the procedure. 11 12 1) Pre-surgical procedures: After reception of the rats, they are transferred 2 per cage 13 and housed in animal facility. They are given pain-killers in specialized gels prior to 14 surgery.15 2) Surgery (TIVAP implantation): TIVAP is implanted subcutaneously with catheter 16 inserted into the jugular vein.

  17

3)29 6 )3311

 6 Postsurgical care: Animals are allowed to recover from surgery before injecting 18 bacteria in the port. During this time of recovery, the patency of TIVAP is maintained by 19 "flush and draw" technique to mimic clinical TIVAP use, and the temperature and weight 20 of the animals are regularly monitored. 21 4) Bacterial challenge: Four days post-surgery, TIVAP is contaminated via controlled 22 bacterial inoculation through the port by bacteria that are allowed to adhere to the device 23 surface (internal) for certain duration of time before the inoculum is removed. 24 5) Post challenge care and monitoring: The biofilm is allowed to form on the device up to 25 day1, day4, day10 or even up to 120 days, depending on the question addressed. Blood 26 sampling is done at day 0 (before inoculation to ensure TIVAP sterility); day4 and day8 27 to analyze the TIVAP mediated infection. Animals are monitored for the clinical 28 symptoms. Euthanasia and sampling: On the day of experiment termination, animals are 30 euthanized and the TIVAP and organs are aseptically removed for analysis. All the described animal work was done in the Institute Pasteur animal facilities, which 34 are accredited by the French Ministry of Agriculture to perform experiments on live 35 rodents (accreditations A75-15 27, issued 12 November 2004, and A75-15 04, issued 22 May 2008). Work on animals was performed in compliance with French and European 1 regulations on care and protection of laboratory animals (European Commission 2 directive 2010/63; French law 2013-118, 06th February, 2013). The protocols used in 3 this study were approved by the ethics committee of "Paris Centre et Sud N°59" 4 (reference 2012-0045). All methods are performed in a 'Biosafety Level-2' facility, 5 containing a Class II biosafety cabinet. Aseptically collected samples (TIVAP, organs or 6 blood) from animals can be processed in the main laboratory inside a Class II biosafety 7 cabinet to maintain sterility. The animal room is cleaned with disinfectant every day until 8 the end of a study. Proper controls must be used in the study. For each study, uninfected animal control 12 with implanted TIVAP should be used as biofilm negative controls. For testing 13 antimicrobials, 1X PBS or other appropriate reagent control must be used to compare 14 the efficacy of the tested compounds. to sedate rat and reduce discomfort during surgery as well as 18 during follow-up procedures. For example, rats treated with cyclophosphamide have 19 different susceptibilities to anesthesia (isoflurane) induced hypoxia; therefore, empirical 20 determination of chemical anesthesia and/or isoflurane dosages is required. We have 21 optimized ketamine, xylazine and acepromazine mix (7:2:1, v/v) injected intraperitoneally 22 to 500 µL cocktail/300grams of rat weight. Acepromazine keeps the rats calm and allows 23 additional injections during the surgery, if needed. Isoflurane concentration for healthy 24 rats was optimized to 3% for the induction of anesthesia and maintenance of the 25 anesthetic state at an oxygen flow-rate of 0.8 L/min and air flow-rate of 0.4 L/min to get a 26 total debit of 1.2. Isoflurane concentration for immunosuppressed rats was optimized to 27 1.5%-2.0% for the induction of anesthesia and 1.5% for maintenance of the anesthetic 28 state at an oxygen flow-rate of 0.6 L/min and air flow-rate of 0.4 L/min to get a total debit 29 of 1.0 L/min. 30 31 Critical step: Isoflurane is an inhalation anesthetics found under several trade names 32 such as aerrane, floran, florane, isothane etc. It provides a quick, easy and long lasting 33 effect when provided continuously to the research animals. Prolonged exposures in 34 animals need to be regulated and monitored closely to prevent death. Injectable 35 anesthetics, such as ketamine-xylazine-acepromazine mixture can be used for long-term (up to 45 min) sedation of rats. Ketamine and acepromazine are controlled 1 substances, which require secured storage and proper records of usage.

2CAUTION:

  Isoflurane is a halogenated ether. It is a colorless liquid anesthetic with a 3 pungent odor. High doses of isoflurane exposure in research animals have been 4 demonstrated to have fetal toxic effects. Pregnant women are recommended not to use 5 it unless other means of anesthesia are not available and, in such case, special masks 6

33

  50µL per TIVAP. The dead volume of the port is 250µL. This insures that no bacteria are 34 flushed into the blood stream at the time of inoculation.

5 275 - 8 1B)

 2758 300grams and are acclimatized to a 12 h day/night cycle for one week prior to use 6 with commercial feed / tap water ad libitum. (TIMING: Responsibility of Animal Facility) 7 Day -5, an electronic chip is inserted in the flank region of rats to monitor the 9 temperature during course of the study. The weight of rats is also monitored on regular 10 basis. (TIMING: 10 min per rat) 11 12 1C) Day -2, all the rats are fed with Medigel-sucralose (1cup (56gm) per cage per day) 13 without ibuprofen* for rats to habituate to mixture followed by Medigel+ibuprofen gel on 14 day -1. (TIMING: 10 min per rat)

3 2D)

 3 Shaving (TIMING: 3-4 minutes per rat) 25 Once the rat is fully unconscious, closely shave dorsal side of the rat with an electric 26 clipper to remove hair from the neck up to lower end of the thoracic vertebrae and from 27 left to right end of the body (Figure 2). Then, turn the rat on its back and shave the 28 neckline carefully without cutting the skin. Remove any excess hair with the help of a 29 clean paper towel. 30 Critical step: If the rat is not completely asleep, any procedure including shaving can be 31 stressful leading to death of rat during the procedure.32 33 2C) Disinfection: (TIMING 5 minutes per rat)34Transfer the rat on a sterile sheet. The shaved area (both dorsal and ventral sides) as 35 well as surrounding area is gently cleaned in 2 steps: i) wash with betadine soap using a 36 sterile gauze pad or any other sterile pad of absorbent material; ii) disinfect with betadine 1 solution. Repeat the cycle 3 times.2 Preparation for surgery: (TIMING ~15 minutes)

72D. 2 )

 2 On the sheet, place on left corner a pair of Scissors, hemostats (2), spring 8 scissors, fine forceps (2), 2 pieces of silk thread around 20 cm long, curved forceps, 9 sterile gloves, TIVAP (with its kit open), scalpel and on the right hand side keep sterile 10 gauze pad, a syringe with 1X PBS (50mL, from the TIVAP kit), vicryl suture, 11 monofilament suture, 1 mL syringe filled with 1X PBS with Huber needle (from the

  17

182E) 27 2E. 4 :172E. 8 :

 2748 Surgery: (TIMING ~30-45 minutes per rat, Figure 2 and Figure 3) 19 2E.1: Once the rat is disinfected, place the rat on the surgical sheet with the dorsal 20 side up and tail away from you. Take some 70% ethanol and wipe the body of the 21 rat once more. Wear the sterile gloves and avoid touching any surroundings. 22 2E.2: Make an incision (~1.5cm) on dorsal line at the upper end of thoracic 23 vertebrae with a scalpel. 24 2E.3: Using a scissor, make a subcutaneous pocket inside the incision by opening 25 & closing the scissor slowly. Make the pocket big enough to easily insert the port of 26 TIVAP. Take the port and insert the pointed end first while pushing the wider part 28 with your thumbs. Once inserted, secure the TIVAP by suturing (using a hemostat 29 to hold the suture and a blunt forceps) inside of the skin with vicryl, 3.0, non-30 absorbable suture.31 2E.5: Insert Huber needle into the port by puncturing the septum and gently turn 32 the rat on its back. Place a pad of gauze pad wet in 1X PBS. 33 Critical step: It is important to keep a wet pad of gauze pad on the incision 34 throughout the process to prevent its dehydration. 2E.6: Make an incision about 1-1.5cm in the neck on right hand side with scalpel. Gently try to remove the muscles with a fine forceps to expose the vein. Keep the 2 area wet and clean with 1X PBS.3 2E.7: Locating the vein: Left Jugular vein is situated superficially under the salivary 4 glands and it forms "inverted Y" junction with the right jugular vein, tail of "inverted 5 Y" joining the subclavian vein. Keep the area and veins wet with 1X PBS, all the 6 time. Critical step: It is important to keep the vein wet because if dried it will lose its 8 flexibility and it will be difficult to make a cut in the vein as well as tough to insert 9 the catheter in vein. 10 Using a curved forceps pass two black silk threads under the external jugular 11 vein. Clamp one on the proximal end (to tie the incised vein) and another on the12 distal end (to secure the catheter inside the external jugular vein) of the jugular 13 vein with hemostats.14 2E.9: Then, try to clean the area surrounding the vein and remove any extra 15 tissues that might be attached to jugular vein. Keep the vein and surrounding area 16 wet with 1X PBS.

  17

2E. 10 :

 10 After exposing the vein, slightly tilt the rat (gently) and insert the blunt end 18 of tunneling rod (provided in the TIVAP kit) from the dorsal side and push out from 19 the ventral incision in the neck region. On the pointed end put on the catheter and 20 pull it along the tunneling rod on ventral side so as to have catheter tunneled under 21 the skin ready to be inserted in the jugular vein.

Step 4 :

 4 Clinical signs: Monitor the clinical signs such as local inflammation around the 17 port, weight loss and fever. If clinical signs are not normal then euthanize the rat. 18 Critical step: Local inflammation can be due to several reasons such as forced 19 injection through the port, infection or too much local injury due to surgery. Bacterial inoculation and monitoring of biofilm formation using 23 bioluminescence: Day +4 (TIMING 30 minutes per rat) (see Figure 4) 24 4A) Bacteria preparation: One day before bacterial challenge, start overnight culture of 25 bioluminescent E. coli 55989 or P. aeruginosa in Lysogeny broth and S. aureus (Xen36, 26 Xen30 or Xen31) or S. epidermidis Xen43 in Tryptic soy agar broth at 37°C shaking at 5mL culture at 7500rpm for 10 minutes to pellet bacteria and re-suspend in 30 5mL 1X PBS. Wash the culture twice in 5mL 1X PBS. Finally, resuspend bacteria in 5mL 31 1X PBS and measure optical density at 600nm (OD600nm).

5 4E) 1 4J)

 51 the bacterial inoculum so as to inject 10 4 CFU/50µL for E. coli, 10 6 CFU/50µL 34 for P. aeruginosa & S. aureus Xen36 and 10 8 CFU/50µL for S. epidermidis Xen43.35 364D) Record the weight and temperature of rat before anesthesia. Clinical symptoms 1 such as local inflammation at the site of port or in the neck region, any other abnormality 2 such as breathing trouble, weight loss and fever must be recorded. Any sick rat must not 3 be included in the study and should be euthanized.4 Anesthetize the rat as described under "flush and draw" method (see step 3B). 6 Meanwhile, spread the sterile sheet in the laminar air flow. Keep betadine solution, 70% 7 ethanol, sterile gauze pads, 50µL bacterial inoculum in 1mL tuberculin graduated 8 syringe, 1mL syringe attached with Huber needle and filled with 1X PBS, 1mL syringe 9 filled with 300µL heparin. 10 11 4F) Anesthetized rat is transferred to the dark box of IVIS imaging system with a charged 12 coupled device (CCD) camera (Xenogen Corporation, Alameda, CA, USA) for monitoring 13 bioluminescence before bacterial challenge. 14 15 4G) After imaging, transfer rat to the hood for infection. Place rat with its nose inside the 16 nose piece and tail away from you. Clean the skin on and surrounding the port with 17 betadine solution and 70% ethanol and allow to air dry for 4-5 minutes. Gently insert the 18 Huber needle connected to a 2mL syringe filled with 1X PBS into the port by puncturing 19 silicon septum of the port. Slowly turn rat on its back keeping the needle inside the port. 20 Slightly inject about 50µL-100µL 1X PBS and then check blood reflux by slowly pulling 21 the piston of the syringe. Remove the syringe keeping the Huber needle inside the port 22 and replace with a syringe to withdraw 150µL blood for analysis. Replace this syringe 23 also with another one containing bacterial inoculum. Slowly push inoculum into the port.24Normally this should take 2 minutes.25Critical step: If the TIVAP is not patent, do not use it for the study. The blood sample is 26 important to check contamination of TIVAP during the post-surgical maintenance. Care 27 should be taken while taking out the needle out of the port to prevent injecting yourself 28 with bacteria. This procedure is always done under the hood if using Class II pathogens.29Injection of inoculum must be very slow to avoid flushing bacteria into the stream. 30 31 4H) After bacterial challenge, rat is again transferred to the dark box of IVIS imaging 32 system with a charged-coupled device (CCD) camera (Xenogen Corporation, Alameda, 33 CA, USA) for monitoring bioluminescence. The rats (with 1X PBS) are used as 34 uninfected controls. 35 36 4I) Rat is then carefully placed back into the cage. Label the cage as Class II. 20 Removal of planktonic bacteria: 3 h after bacterial inoculation, the planktonic bacteria 2 are removed leaving only the adhered bacteria to form biofilm. For this, repeat the 3 anesthesia procedure as above. Insert a Huber needle attached to a 1mL syringe 4 containing 500µL 1X PBS and flush 50-100µL in the TIVAP very slowly. Replace the 5 syringe with new 1mL syringe to withdraw about 300uL blood. Remove this syringe with 6

5 : 28 5C)

 528 Post challenge care and monitoring: (TIMING: 30 minutes per rat) 9 5A) Monitoring Clinical symptoms: Carefully monitor/record weight loss, fever, diet loss 10 or any other abnormal behavior. Any rat with temperature above 39°C +/-0.5 or below 11 34°C +/-0.5 for continuous 3 days must be euthanized. The sick rats must be 12 euthanized. 13 14 5B) Bioluminescence for biofilm development and pathogenesis (see Figure 4): Biofilm 15 formation inside the lumen of the TIVAP and associated infection is measured as a 16 function of bioluminescence. Anesthetized rat (either isoflurane or chemical) is 17 transferred to the dark box of IVIS imaging system with a charged coupled device (CCD) 18 camera (Xenogen Corporation, Alameda, CA, USA) for monitoring bioluminescence. 19 Length of exposure can vary depending on the bacteria and the expected infection. The 20 exposure time can vary depending upon bacteria and infection. Normally, 1 minute 21 exposure is sufficient for E. coli and P. aeruginosa but for S. aureus Xen36 exposure can 22 be about 2-4 minutes. Control rats are exposed for the same or more time compared to 23 test rats. 24 Critical step: Exposing control rat is important to evaluate and measure background 25 coming from different material such as animal feed or reagents used. It is important to 26 use at least 4 control rats per study for statistics. 27 Blood sampling: Blood sampling can be done on day 4 and day 8 post-infection to 29 monitor the blood stream infection and associated changes in host factors such as 30 cytokine analysis. Blood (150µL) can be drawn either from the caudal (tail) vein or by 31 retro orbital plexus puncture.

5

  Pasteur sterile pipette to gently and slowly puncturing the orbital plexus. While firmly 6 holding the Pasteur pipette near the sinus, with gentle rotating movements insert the 7 tube through the membrane. Keep rotating the tube through the orbit until the blood 8 starts to flow. Collect the appropriate volume of blood and transfer to a collection tube 9 containing anti-coagulant (for viable bacteria count, estimation of immune cell 10 population) or tube without anti-coagulant (for cytokine analysis).

  Extraction and quantification of biofilm bacteria from TIVAP: (TIMING: ~10-15 min 28 per rat) All the procedure is done under the laminar air flow (P2 lab).

296E. 1 )

 1 TIVAP are carefully wiped with 70% EtOH before extracting intraluminal biofilm 30 bacteria to avoid contaminant.

356E. 4 ) 4 6E. 6 )

 446 Cells attached to the titanium body of the port are scratched in 100 mL 1X PBS 36 and transferred to the same tube as the septum. 6E.5) Biofilm that formed on the septum and in the lumen of catheter is extracted by 1 vigorously vortexing the tubes for 1 min, followed by transferring them to an ultrasonic 2 water bath (NEYtech Ultrasonik, 44-48 Khz) for 5 min and a second vortexing for 1 3 minute. The bacterial suspension is then diluted serially, plated on agar plates and 5 incubated at 37°C for colony counts. CFU/mL and bioluminescent signals (ROI, 6 p/S/cm 2 /sr) are plotted together for correlation.

  pocket infection: (TIMING ~20 min per rat) 10 supp1A) Any inflammation around the port after injecting the bacterial inoculum in the 11 port indicates a pocket infection.12 supp1B) From euthanized rat, carefully remove the port from the pocket by opening the 13 incision made at the time of surgery. Cut the sutures used for securing the port in the 14 pocket. 15 supp1C) Image the rat in the dark box of IVIS imaging system with a charged coupled 16 device (CCD) camera (Xenogen Corporation, Alameda, CA, USA) for monitoring 17 bioluminescence and evaluate the bacterial load in the pocket.18 supp1D) Remove all the pus formed in the pocket for CFU estimation.

19supp1E)

  Plate the serial dilutions for E. coli and P. aeruginosa on LB and for S. aureus 20 infection: (TIMING ~10 min per rat) 23 supp2A) S. aureus Xen36 is used to check the possibility of TIVAP colonization through 24 the venous system.25 supp2B) Overnight grown S. aureus Xen36 culture is used as inoculum. An inoculum of 26 5 X 10 8 cells of S. aureus Xen36 is centrifuged, washed and resuspended in 500µL 1X 27 PBS. 28 supp2C) Anesthetize the rat as described above. In case using chemical anesthesia, 29 inject only 200µL intraperitoneally as the procedure takes only 10 minutes for injection.30 supp2D) Clean the tail with 70% ethanol and rub the tail with a sterile cotton swab to 31 remove the dead scales. Approximately 5cm above the tail end, inject bacteria into the 32 bloodstream of rat (already containing implanted TIVAP) through the lateral tail vein.

33supp2E)

  Monitor rat before and after injecting bacteria for bioluminescence using IVIS-

34 5 supp3)

 5 100 imaging system to check infection and colonization.35supp2F) 3-days after the injection euthanize rats as described earlier in the protocol. supp2G) TIVAP is removed aseptically to confirm the colonization by bioluminescence 1 and plating CFU/mL. 2 supp2H) 1-cm tip of the catheter is plated for CFU enumeration as the bacteria colonized 3 only the tip of catheter.4 Immunosuppression of rats: (TIMING ~15-20 min per rat) 6 supp3A) Rats are injected intraperitoneally with cyclophosphamide.

  7

8 more

 8 lumen of the catheter and face of the septum inside the port are used to visualize 5 are stored at 4°C until sent for microscopy to the core facility / experts (not

19(

  30mg/kg, for Gram-negative bacteria) subcutaneous injections42,43 . The 4-day-old biofilm 20 formed inside the implanted TIVAP is locked (200µL) with the above-discussed antibiotics 21 and monitored for biofilm clearance by measuring bioluminescence. Two types of regimen 22 are followed for lock therapy: first is a 5-day lock regimen during which the old lock is 23 replaced by a new one every 24 h for 5 days in conjunction with systemic treatment for 5 24 days. We also assess 1 day lock regimen with a single instillation dwelling for 7 days in 25 conjunction with 1 day of systemic treatment. Rats with a colonized TIVAP but receiving PBS 26 lock are used as controls. 27 28 5A) Anesthetize infected rat and measure the bioluminescence as described above after 4 29 days of infection in TIVAP. 30 31 5B) Rat is transferred in the hood for instilling antibiotic solution in the TIVAP. Instill 200 µL 32 antibiotic solutions in the port slowly. Vancomycin or gentamicin for Gram positive and Gram 33 negative bacteria respectively is injected intraperitoneally to prevent systemic infection due to 34 flushing of the lock into the blood circulation. 35 36 5C) Replace the lock every 24 h. the catheter out of the vein slowly. Press the vein with a wet gauze pad to prevent blood loss and prevent the hydration of vein. Check the length of the catheter and cut it to the right length. If the catheter is shorter, than try to change the position of the port so that some length can be gained by reducing the distance between the port and the vein (in this case one may have to optimize the dead volume to avoid flushing of bacteria into the blood stream at the time of challenge). b) Pull out the catheter as above and make the right cut and reinsert it. , it is not advisable to make a bigger slant for the ease of insertion but one can try to lift the rats from the back slight to open the channel or try to gently and slightly move the catheter without pulling it out of the vein. b) If the catheter has slipped out of the vein, rat must be excluded from the study. properties may change from lot to another. So, try changing the lot used. d) Contamination from the rat flora can prevent biofilm formation of your strain. Disinfect the skin of the rat properly to avoid any external contamination.

13

  Reduced signals were observed from day 8 onwards, indicating restriction of biofilms to 14 TIVAP colonization (Figure5)27 . These results can be correlated to the bloodstream 15 infection as indicated by presence of bacteria on day 4 whereas clearance of bacteria 16 from the peripheral blood by day 8. Thus, this model allows non-invasive study of chronic 17 immunosuppressed rats, severe pathology can be seen as indicated 20 by weight loss, fever and high bioluminescence as function of systemic biofilm infection 21 leading to death of animals by day 3 (Figure6)27 . The blood and organ samples show 22 high bacterial load compared to immune-competent rats. case of lock therapy experiments, the absence of bioluminescence may be 25 observed just after 1 instillation of antibiotic solution, such as in case of 26 gentamicin+EDTA lock. On contrary, this bioluminescence signals indicating presence of 27 persistent bacteria in the catheter may be seen as in case of gentamicin, EDTA, L-28 arginine alone (Figure7)28 . Lock therapy may lead to bloodstream and systemic 29 infection in some rats. Thus, use of systemic treatment in conjunction with lock therapy is 30 required.

7 8 Figure 2 .

 82 Figure 2. TIVAP surgical implantation. TIVAP were surgically implanted in CD/SD

11

  in figure correspond to steps described in the procedure under surgery section. (2A) Rat 12 was briefly kept in an isoflurane box to calm down and injected intraperitoneally with a 13 chemical anesthesia mixture to complete sedation and analgesia before starting the 14 procedure. (2B.1 -2B.2) Rat was shaved on dorsal side from the neck to the lower end 15 of thoracic vertebrae and along the neck line of ventral side. (2C) Disinfection of skin 16 using betadine soap and solution. (2D) Pre-surgical preparations. Instruments and 17 objects required during the surgery kept on a sterile sheet in the laminar air flow. (2E.2) 18 Incision was made along the dorsal line at the upper thoracic vertebrae. (2E.4.1 -2E.4.2 19 1 -2E.4.3) TIVAP inserted in the subcutaneous pocket and secured by sutures. (2E.5.1) 20 Huber needle inserted in the port. (2E.5.2) Rat flipped on its back. (2E.6) 1-1.5cm

3 4Figure 7 .

 37 Figure 7. In vivo lock therapy against Staphylococcus aureus biofilm in the

8 aureus. 22 Figure 8 .

 8228 photon emissions. (A) Control rats with PBS lock. (B) 5 mg/mL gentamicin lock. (C) 30

Equipments: 2 Equipments (number needed for the study) Catalogue number Manufacturer

	1 1	Buprecare ® (Ibuprofen) TIVAP, Polysite 2000 micro, 5F		2105 ISP	Axience, France Perouse Biotec
	2 2	MATERIALS Cyclophosphamide monohydrate Cell strainer 70µm Nylon PROCEDURE		C0768-5G 3523350	Sigma Aldrich medical BD Falcon
	3 3	Reagents: Sodium Cacodylate Trihydrate Sterile champ T25 digital ULTRA-TURRAX ® homogenizer	C0250-100G CSO-02NT 0010001502	Sigma Aldrich LCH Ika, France
	4	Ruthenium red Sterile gloves (size 7-8, medium) NEYtech Ultrasonik, 44-48 Khz		R2751-1G STP641	Sigma Aldrich LCH NEYtech
	1	Product Glutaraldehyde 25% Sterile gauze pad		Reference G5882 CNST-470	Manufacturer Sigma Aldrich LCH
	1 2	Sterile 1X Phosphate buffered Saline Huber needle, straight 7/10, 22G, 30mm, box	10010-023 512507	GIBCO Perouse
		(PBS) of 50			Medical
		Sterile water Respiratory Mask, FFP2		15230-071 19-130-4825	GIBCO Fisher
		Kanamycin		B5264-1G	Sigma Aldrich Scientific
	10	Cefazolin Charged-coupled device CCD Camera (IVIS Sterile gown	C5020-98000622	Sigma Aldrich Xenogen Barrier,
		100)		500MG	Corporation, France
		Vancomycin Temperature Controller, with rectal probe	94747-5G TCAT-2LV	Sigma Aldrich Alameda, Physitemp
		Gentamicin		G1914-5G	Sigma Aldrich CA, USA Instruments
		Lysogeny Broth (LB) Vet abc		12795-027 ABC vet 2.0	Invitrogen SCIL, Inc, USA
		L-arginine Thermocage, animal warming system	A5006-100G MK3	Sigma Aldrich Germany Datestand
		Agar degranulated Surgical hood		214530 FASTER	DIFCO Faster-air, Ltd.,
		Tryptic Soy broth (TSB)		211825 FLOWFAST	DIFCO France Manchester,
		Tryptic soy agar (TSA)		236950 H18	DIFCO UK
		Ethanol, 70% (made from absolute Laminar air flow IPTT-300, Electronic chip introducer and chip	34935-1L Faster BV 11059	Sigma Aldrich Faster-air, PLEXX,
		alcohol)		BH2006	France Europe
		Sterile Heparin, 500IU/mL, 5mL bottle, Hot bead sterilizer Wireless Reader with round IMI probe	512507 18000-50 DAS-7006R	Sanofi Aventis Fine Science PLEXX,
		Ketamine, Imalgen1000		IMA004	Merial SAS Lyon, Fr Tools (FST) Europe
		Xylazine, (Rompun™2%) Beads Mozer Max45 clipper		ROM001 18000-51 1245-0066	Bayer Healthcare, FST Moser
		Micro spring scissors (1)		15007-08	Germany FST animalline
		Acepromazine Fine scissors (1) Isoflurane vaporizer, TAG (Injectable),	Calmivet	CAL226 14502-14 TAG1100	Sanofi FST TEM	Aventis,
		Solution Fine forceps (2)		11245-30	Lure, FR FST (Lormont,
		Betadine gel 10%, tube of 100g Blunt forceps (1)		11000-14	Meda Pharma FST France)
		Anesderm 5%, tube of 5g Hemostats (2) Conical tubes , 50mL		Pharmacy 130008-12 14-432-22	Pierre FST Fisher	Fabre,
		Silk Thread, 22.5 meters		18020-30	France FST scientific
		Betadine solution, bottle of 120mL Curved forceps Conical tubes , 15mL	91197-00 14-959-70C	Vetoquinol FST Fisher
		Eye drops, Ocrygel ® Suture Vicryl 3-0 Polyglactin absorbable, box	843TVN V497H	Ethicon scientific
		Gel, medigelsucralose 56gm of 36 Microtubes		TPP2270 05-402-24B	Fisher Scientific Fisher
		Vikron ® 1% Suture Dafilon 3/0 Polyamide non-absorbable,	C0935239	Reltyon B Braun scientific
		Sodium pentobarbital, Dolethal box of 36 GentleMACS™ Octo Dissociator		6847542 130-095-937	Alcyon France Miltenyi
		Isoflurane, IsoVet 1000mg/g inhalation Scalpel	IsoVet 0510	Imaxio, France Swann-Biotec
		vapour (Schering-Plough) GentleMACS™ M tubes		1000mg/g 130-096-335	Morton Miltenyi
						10 14

  Na-Cacodylate is a derivative of arsenic. It is highly toxic when inhaled, ingested,

	8	CAUTION: cyclophosphamide is toxic and immunosuppressant. Cyclophosphamide is an
	9	antineoplastic (anti-cancer) drug categorized as an alkylating agent. Its side effects depend
	10	upon dosage such as lowered blood counts, sterility in males and females, pregnancy
	11	defects and or discoloration of skin and nails. Using the chemical hood and gloves is highly
	12	recommended when using this compound. For detailed literature on side effects, consult:
	20
	21	Critical step: An optimized inoculum dose of 10 2 CFU/ 50µL 1X PBS for all bacterial strains
	22	(higher inoculum doses lead to the death of animals overnight) is used for TIVAP
	23	contamination and is confirmed by plating for CFU/mL. Control catheterized and
	24	immunosuppressed rats receive 50µL 1X PBS only.
	25	supp3C) Prior to inoculation of clinical strains, all rats are checked for the absence of
	26	infections as for immune-competent rats.
	27
	28	4) Electron microscopy: (TIMING ~30 min per rat)
	29	4A) After aseptic removal of colonized TIVAP from rats, 1 cm catheter of the catheter tip is
	30	cut and the septum is dissected from the port using a scalpel.
	31
	32	4B) Septum and catheter pieces are washed twice in cacodylate solution prepared with
	33	gloves and under a chemical hood (0.07 M: 10.5mL 0.2M Na-cacodylate pH 7.4 + 19.5 mL

http://www.ncbi.nlm.nih.gov/pubmed/?term=Cyclophosphamide+side+effects 13 supp3B) Dose and regimen of cyclophosphamide delivery are optimized by estimating blood 14 total leukocyte count as determined using an animal blood cell counter (Vet abc, SCIL,

15

Germany ABC vet 2.0, Germany). Furthermore, immunosuppressed status of rats is 16 indicated by the physical state of rats such as lack of appetite, reduced weight and loss of 17 hair. One hundred mg/kg body weight of cyclophosphamide was finally selected for giving 18 intraperitoneal injections to rats (n= 4 for each bacterial strain) on day -4 and 50 mg/kg on 19 day -1 of inoculation. sterile D/W) and then fixed in EM fixative solution (for example, a mixture v/v/v of 6% 34 glutaraldehyde, 0.15% Ruthenium red, 0.2M Na-cacodylate pH 7.4).

CAUTION:

Table 2

 2

	2	. TROUBLE SHOOTING
	3
	4

Table 1 : Lock therapy concentration (mg/mL) LEGENDS TO FIGURES: 1 2 Figure 1. Schematic representation of the different steps of the in vivo model of 3 totally implantable venous access port related infection.

 1 Steps in black boxes

	31
	32
	33	4.	In addition to biofilm pathogenesis, molecular mechanisms associated with
	34	biofilm can be studied 29 . It was shown using mutants of a bioluminescent variant of E.
	35	coli 55989 that lipid A palmitoylation did not affect the bacterial adherence to catheters
		but enhanced the in vivo biofilm survival. Moreover, the palmitoylation lead to reduced

Biofilm formation in TIVAP was confirmed by scanning electron 16 microscopy

  French law 2013-118, 06th February, 2013). The protocols used in this study were 1 approved by the ethics committee of "Paris Centre et Sud N°59" (reference 2012-0045).

	1	and protection of laboratory animals (European Commission directive 2010/63; French
	2 2	law 2013-118, 06th February, 2013). The protocols used in this study were approved by
	3	the ethics committee of "Paris Centre et Sud N°59" (reference 2012-0045).
	4	Figure 4: Bacterial inoculation and monitoring of biofilm formation using
	5	bioluminescence. The bacterial inoculum is directly injected through the septum of the
	6	TIVAP using a Huber needle and biofilm development is monitored using a CCD camera
	7	imaging system allowing to measure bioluminescence. Below is shown an example of
	8	the images obtained for the colonization of the TIVAP by a bioluminescent clinically
	9	relevant strain of P.aeruginosa and showing the progressive biofilm development within
	10	the TIVAP. Work on animals was performed in compliance with French and European
	11	regulations on care and protection of laboratory animals (European Commission
	12	directive 2010/63; French law 2013-118, 06th February, 2013). The protocols used in
	13	this study were approved by the ethics committee of "Paris Centre et Sud N°59"
	14	(reference 2012-0045).
	15
	21 22 23 Figure 5. 23 Incision in neck region (2E.7.1 -2E.7.2) Jugular vein exposed. Work on animals was performed in compliance with French and European regulations on care and protection
	24 24	of laboratory animals (European Commission directive 2010/63; French law 2013-118, animals was performed in compliance with French and European regulations on care
	25 25	06th February, 2013). The protocols used in this study were approved by the ethics and protection of laboratory animals (European Commission directive 2010/63; French
	26 26	committee of "Paris Centre et Sud N°59" (reference 2012-0045). law 2013-118, 06th February, 2013). The protocols used in this study were approved by
	27 27	the ethics committee of "Paris Centre et Sud N°59" (reference 2012-0045).
	28 28	Figure 3. TIVAP surgical implantation (contd.). (2E.8) Cotton threads inserted under
	29 29	the jugular vein to secure catheter. (2E.10) Tunneling rod inserted for passing the Figure 6. Biofilm led to lethal infection in immunosuppressed rats. (A) TIVAP
	30 30	catheter under the skin from dorsal to ventral side. (2E.11) Catheter cut at a slant (inside implanted and cyclophosphamide-treated rats were injected with 10 2 CFU in 100mL of P.
	31 31	caption showing the correct cut). (2E.13) Jugular vein incised using micro spring aeruginosa into the port of TIVAP and photon emission was monitored up to day 3 to
	32 32	scissors. (2E.14) Dilation of jugular vein using forceps. (2E.14.1 to 2E.14. 5) Catheter evaluate biofilm formation and associated infection. (B) Bacterial load from different
	33	inserted in the jugular vein. (2E.15) Flush and draw blood to check patency of TIVAP. organs aseptically removed from dead animals was analyzed, organs were
	36

33

(2E.17.1 -2E.17.2) Surgical wounds closed by suturing ventral and dorsal incisions. 34 Work on animals was performed in compliance with French and European regulations on 35 care and protection of laboratory animals (European Commission directive 2010/63; (SEM). TIVAPs implanted in rats and inoculated with S. aureus, P.

17

aeruginosa or E. coli were harvest on day 5 post-infection and analyzed with scanning 18 electron microscopy. Arrows represent bacteria in biofilm; arrowheads, blood cells. (A) 19 TIVAP inoculated with S. aureus, (B) TIVAP inoculated with P. aeruginosa, (C) TIVAP 20 inoculated with E. coli. (E) Scanning electron microscopy to show P. aeruginosa biofilm 21 growing inside the lumen of implanted catheter with typical biofilm micro colonies. (F-G) 22 Magnified view: a P. aeruginosa micro colony showing typical biofilm structures. Work on homogenized and were plated on LB agar for viable counts per mL. CFU results are 34 means +/-standard deviations. Number of rats (n) used in the experiment, n = 4. Work 35 on animals was performed in compliance with French and European regulations on care

  Percival, S. L., Suleman, L., Vuotto, C. & Donelli, G. Healthcare-associated 3 infections, medical devices and biofilms: risk, tolerance and control. Journal of 4 medical microbiology 64, 323-334, doi:10.1099/jmm.0.000032 (2015). . A. et al. Clinical practice guidelines for the diagnosis and management of 13 intravascular catheter-related infection: 2009 Update by the Infectious Diseases 14 Society of America. Clin Infect Dis 49, 1-45, doi:10.1086/599376 (2009). Perez-Molina, J. A. & Munoz, P. Report of ESGNI-001 and ESGN2-002 16 studies. Bloodstream infections in Europe. Clinical Microbiology and Infection 5, 2S1et al. The Calgary Biofilm Device: new technology for rapid determination of 19 antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol 37, 1771-1776 (1999). 20 7 Coenye, T. & Nelis, H. J. In vitro and in vivo model systems to study microbial biofilm :10.1016/j.mimet.2010.08.018 (2010). 23 34 Mussard, W., Kebir, N., Kriegel, I., Esteve, M. & Semetey, V. Facile and efficient 1 control of bioadhesion on poly(dimethylsiloxane) by using a biomimetic approach. Angewandte Chemie 50, 10871-10874, doi:10.1002/anie.201101029 (2011). 3 35 van Rooden, C. J. et al. Infectious complications of central venous catheters increase 4 the risk of catheter-related thrombosis in hematology patients: a prospective study. Journal of clinical oncology : official journal of the American Society of Clinical 6 Oncology 23, 2655-2660, doi:10.1200/JCO.2005.05.002 (2005). Control of Pseudomonas aeruginosa in the lung requires the 8 recognition of either lipopolysaccharide or flagellin. Journal of immunology 181, 586-Gounon, P. & Le Bouguenec, C. Identification of an aggregative adhesion 11 fimbria (AAF) type III-encoding operon in enteroaggregative Escherichia coli as a 12 sensitive probe for detecting the AAF-encoding operon family. Infection and immunity . L., Thomas, L., Goussard, S., Branchini, B. R. & Grillot-Courvalin, C. In 15 vivo bioluminescence imaging for the study of intestinal colonization by Escherichia 16 coli in mice. Appl Environ Microbiol 76, 264-274, doi:10.1128/AEM.01686-09 (2010). 17 39 McGuill, M. W. & Rowan, A. N. Biological Effects of Blood Loss: Implications for 18 Sampling Volumes and Techniques. ILAR News 31, 5-20 (1989). 19 40 Removal of blood from laboratory mammals and birds. First report of the 20 BVA/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Laboratory

	1
	2 2
		1
	5
	5 6 7	2	Hoiby, N. et al. ESCMID guideline for the diagnosis and treatment of biofilm infections
	2014. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 21 Suppl 1, S1-25, Ramphal, R. et al. 9 7 36 8 doi:10.1016/j.cmi.2014.10.024 (2015). 9 592 (2008). 10
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	34

doi22 41 van Herck, H. et al. Orbital sinus blood sampling in rats as performed by different 23 animal technicians: the influence of technique and expertise. Laboratory animals 32, 24 377-386 (1998). 25 42 Van Praagh, A. D. et al. Daptomycin antibiotic lock therapy in a rat model of 26 staphylococcal central venous catheter biofilm infections. Antimicrobial agents and 27 chemotherapy 55, 4081-4089, doi:10.1128/AAC.00147-11 (2011). 28 43 Zuluaga, A. F., Agudelo, M., Cardeno, J. J., Rodriguez, C. A. & Vesga, O.

Critical step: It is possible that TIVAP may be blocked because of biofilm formation and old 1 lock may not be retrievable. In this case flush the old lock with PBS and then instill the fresh 2 lock in the port. Step1: day -6 to day-1 (6 days)

3

Step2: 1-2 days (step 2A to 2E.19: ~55 min per rat per day and step2D: could be done in 4 parallel with step 2A)

5

Step3: 4 days; 3A to 3C: ~10 min per rat per day 6

Step 4A to 4J: 2 days; ~30 min per rat (Inoculum is started a day before the challenge)

7

Step A to 5C ~30 min per rat.

8

Step 5C.1 /5C.2: ~10 min per rat 9

Step 6A to 6E ~45 min per rat