Lakshmanan Kuppusamy

Anand Mahendran

Eric Villemonte De La Clergerie

Modelling Intermolecular Structures and Defining Ambiguity in Gene Sequences using Matrix Insertion-Deletion Systems 1

Keywords: insertion-deletion systems, intermolecular structures, ambiguity, matrix grammars, gene sequences

, a new computing model named Matrix insertion-deletion system has been introduced to model various bio-molecular structures such as hairpin, stem and loop, pseudoknot, attenuator, cloverleaf, dumbbell that occur at intramolecular level. In this paper, we model some of the intermolecular structures such as double strand languages, nick languages, hybrid molecules (with R-loops), holliday structure, replication fork and linear hybridization (ligated) languages using Matrix insertion-deletion system. In [2], the ambiguity in gene sequence was defined as deriving more than one structure for a single gene sequence. Here, we propose a different view of understanding the ambiguity in gene sequences: A gene sequence is obtained by more than one way such that their intermediate sequences are different. We further classify the ambiguity into many levels based on the components axiom, string (order of deletion/insertion) and contexts (order of the used contexts). We notice that some of the inter and intramolecular structures obey the newly defined ambiguity levels.

Introduction

Insertion-deletion systems are introduced to theoretically analyze the insertion and deletions operations that take place in gene sequences. Informally, the insertion and deletion operations of an insertion-deletion system is defined as follows: If a string β is inserted between two parts w 1 and w 2 of a string w 1 w 2 to get w 1 βw 2 , we call the operation as insertion, whereas if a substring α is deleted from a string w 1 αw 2 to get w 1 w 2 , we call the operation as deletion. These systems opened a particular attention in the field of formal languages as the system is not exactly based on rewriting systems.

As DNA and RNA molecules can be considered as a sequence of symbols (i.e., strings) over {a, t, g, c} and {a, u, g, c} respectively, problems that exist in such molecules can be studied using formal grammars. The following example witnesses the correlation between formal grammars and bio-molecular structures. Consider a contextfree language {ww R | w ∈ {a, b} * } where w R is the reverse of w. Consider the following gene sequence gcatgcgcatgc. As the complementary pairs ā = t, t = a, ḡ = c and c = g, the above gene sequence resembles a word in the palindrome language {w wR | w ∈ {a, t, g, c} * }.

There exist some relations between intramolecular gene sequences and natural language constructions such as triple agreements : {a n b n c n | n ≥ 1}, crossed dependencies: {a n b m c n d m | n, m ≥ 1} and copy language: {ww | w ∈ {a, b} * } [1], [START_REF] Searls | The comp. linguistics of biological sequences[END_REF]. In below, we discuss briefly some of the important intra and intermolecular structures that are predominantly available in bio-molecules such as protein, DNA and RNA. Fig. 1 shows the intramolecular structures (a) stem and loop, (b) cloverleaf and (c) dumbbell. Note that these structures can be represented by context-free grammars. However, there are some more structures that occur frequently in bio-molecules which cannot be modelled by context-free grammars. Fig. 2 represents such intramolecular structures (a) pseudoknot and (b) attenuator. In Fig. 1(a) and Fig. 2, the strings are obtained by reading the symbols as per directed dotted lines. The string ugcucaag (refer Fig. 2a) represents the pseudoknot structure, the string agaucuaga (refer Fig. 2b) represents the attenuator structure and they resemble the crossed dependency language and the copy language respectively.

0 0 1 1 0 0 1 1 0 0 1 1 5' C A A U G A C 3' U A U G U C A A C G A B C u u _ R (a) (b) (c)
a g a t c t t g a t c a In RNA, the gene sequences are mostly intermolecular rather than intramolecular. Such intermolecular structures are modelled by cutting the intramolecular secondary structures randomly by leaving some open points such that base pairing can be done at these open points. Fig. 3 represents some of the intermolecular structures (a) double strand languages and (b) nick languages where the cut takes place at arbitrary position is represented by a •. In Fig. 3 S, stands for the non-terminal in their corresponding context-free grammar.

In the last two decades, attempts have been used to model these intra and inter molecular structures by defining new grammar formalisms like crossed-interaction grammar [START_REF] Rivas | The language of RNA: A formal grammar that includes pseudoknots[END_REF], simple linear tree adjoining grammars and extended simple linear tree adjoining grammars [START_REF] Uemura | for RNA structure prediction[END_REF]. In [START_REF] Searls | Formal grammars for intermolecular structures[END_REF], cut grammars, ligation grammars were defined specifically to model intermolecular structures. However, there is no unique grammar system that encapsulates all essential and important (intra/inter) bio-molecular structures. For example double copy language cannot be modelled by a simple linear tree adjoining grammar [START_REF] Uemura | for RNA structure prediction[END_REF]. Very recently, in [START_REF] Kuppusamy | Matrix insertiondeletion systems for bio-molecular structures[END_REF] a new simple and powerful biologically inspired computing model namely Matrix insertion-deletion system has been introduced by combining insertion-deletion system and matrix grammar with a strong motivation to model the above mentioned intramolecular structures in terms of languages. Though, insertiondeletion system itself is computationally complete, and therefore possible to generate any recursive enumerable language, constructing insertion-deletion grammars for some languages (that form bio-molecular structures) is not easy and the procedure to simulate the grammar is very tedious. For example, consider the triple agreement language {a n b n c n | n ≥ 1} that forms the triple stranded DNA. Defining the insertion-deletion grammar is very difficult since we need to make sure that whenever one a is introduced, simultaneously, one b and c must be introduced at the appropriate places, thus the synchronization between a, b and c is taken care. To do this in insertion-deletion system, we need to introduce a new marker near the inserted a and move the marker towards right by passing all a until a b is encountered (for inserting b) and then move further to encounter a c (to introduce a c). Besides, the marker need to be deleted on crossing each symbol. If not constructing this way, we need to identify the Chomsky grammar which generate this language, and convert it to a Kuroda (or suitable) normal form and from that nor-mal form, we can obtain the insertion-deletion grammar using the computational completeness result. This is also not an easy way when compared to the grammar of Matrix insertion-deletion system as we are going to see how simply we can construct a grammar in Matrix insertion-deletion system for such languages and how freely the synchronization is achieved. In [START_REF] Kuppusamy | On representing natural languages and bio-molecular structures using Matrix insertion-deletion systems and its computational completeness[END_REF], the computational completeness of Matrix insertion-deletion system has been analyzed. Interestingly, in [START_REF] Petre | Matrix insertion-deletion systems[END_REF], the same Matrix insertion-deletion system has been introduced independently but without such motivations and its computational completeness has been analyzed. In this paper, we model some of the intermolecular structures that are predominantly available in gene sequences (more precisely in RNA and proteins).

Ambiguity is considered to be one of the important issues not only in natural and programming languages, but also in gene sequences. In [START_REF] Kuppusamy | Matrix insertiondeletion systems for bio-molecular structures[END_REF], the ambiguity in gene sequences are dealt informally by showing that there exists some bio-molecular structures which has the notion of ambiguity. Since the insertion-deletion system can be applied theoretically in DNA processing [START_REF] Gheorghe Pȃun | New Computing Paradigms[END_REF], the ambiguity in DNA processing (which uses the insertion-deletion system) may happen in the following manner. Let W 1 W 2 be a DNA strand and suppose we want to insert W 3 W 4 W 5 between W 1 and W 2 to obtain another DNA strand

W 1 W 3 W 4 W 5 W 2 .
This can be done first by inserting W 3 between W 1 and W 2 , followed by inserting W 4 between W 3 and W 2 , followed by inserting W 5 between W 4 and W 2 . The other sequence would be first by inserting W 5 between W 1 and W 2 , followed by inserting W 4 between W 1 and W 5 , followed by inserting W 3 between W 1 and W 4 . More precisely the derivations are given below: (the underlined string denotes the inserted string) (1)

W 1 W 2 =⇒ W 1 W 3 W 2 =⇒ W 1 W 3 W 4 W 2 =⇒ W 1 W 3 W 4 W 5 W 2 (2) W 1 W 2 =⇒ W 1 W 5 W 2 =⇒ W 1 W 4 W 5 W 2 =⇒ W 1 W 3 W 4 W 5 W 2 .
This shows that ambiguity in gene sequences is also possible (i.e., starting from one sequence we are able to get another sequence in more than one way such that the intermediate sequences are different). With this view of ambiguity, in this paper, we formally define the various levels of ambiguity for Matrix insertion-deletion systems. Study of this concept of ambiguity may be useful while considering inheritance properties and phylogenetic trees [START_REF] Setubal | Introduction to computational molecular biology[END_REF].

Preliminaries

Here, we recall the basic notions which are used in the paper. A finite non-empty set V or Σ is called an alphabet. We denote by V * or Σ * , the free monoid generated by V or Σ, by λ its identity or the empty string, and by V + or Σ + the set V * -{λ} or Σ * -{λ}. The elements of V * or Σ * are called words or strings. For a string w, |w| b denotes number of b in w. The language of DNA (RNA) can be considered over

Σ * DN A(RN A) = {a, t(u), g, c}.
For more details on formal language theory, we refer to [START_REF] Rozenberg | [END_REF].

Next, we look into the basic definitions of insertion-deletion systems. Given an insertion-deletion system γ = (V, T, A, R), where V is an alphabet, T ⊆ V , A is a finite language over V , R is a finite triples of the form (u, α/β, v), where (u, v) ∈ V * × V * , (β, α) ∈ (V + × {λ}) ∪ ({λ} × V + ). The pair (u, v) is called contexts. Insertion rule will be of the form (u, λ/β, v) which means that β is inserted between u and v. Deletion rule will be of the form (u, α/λ, v), which means that α is deleted between u and v. In other words, (u, λ/β, v) corresponds to the rewriting rule uv → uβv, and (u, α/λ, v) corresponds to the rewriting rule uαv → uv.

Consequently, for x, y ∈ V * we can write x =⇒ * y, if y can be obtained from x by using either an insertion rule or a deletion rule which is given as follows: (the down arrow ↓ indicates the position where the string is inserted, the down arrow ⇓ indicates the position where the string is deleted and the underlined string indicates the string deleted/inserted)

1. x = x 1 u ↓ vx 2 , y = x 1 uβvx 2 , for some x 1 , x 2 ∈ V * and (u, λ/β, v) ∈ R. 2. x = x 1 uαvx 2 , y = x 1 u ⇓ vx 2 , for some x 1 , x 2 ∈ V * and (u, α/λ, v) ∈ R.
The language generated by γ is defined by

L(γ) = {w ∈ T * | x =⇒ * w, f or some x ∈ A}
where =⇒ * is the reflexive and transitive closure of the relation =⇒.

Next, we will look into the definition of cut grammars. A cut grammar G = (N, Σ, S, P ) where N is a finite set of non-terminals, Σ is a finite set of terminals, S is a start symbol and P is a finite set of productions in

(N ∪Σ) * N (N ∪Σ) * ×(N ∪Σ∪{♦}) * .
where ♦ is a new symbol called cut symbol not in N or Σ. The language generated by cut grammar is defined as L(G) = {w ∈ (Σ ∪ ♦) * | S =⇒ * w}. Given any string w = w 1 ♦w 2 ♦ . . . ♦w n where w i ∈ Σ * for 1 ≤ i ≤ n, the cut function w = {w 1 , w 2 , . . . , w n } and the uncut function w = w 1 w 2 . . . w n . For a given cut grammar G and start symbol S, the cut language is defined as L(G) = { w ∈ 2 Σ * | S =⇒ * w} and the uncut language L(G) = { w ∈ Σ * | S =⇒ * w}. A ligation grammar is a cut grammar with an additional new symbol ♯. For any w = w 1 ♯w 2 ♯ . . . ♯w n , where w i ∈ (Σ ∪ {♦}) * for each 1 ≤ i ≤ n, the ligate function is defined as w = { w 1 , w 2 , . . . , w n }. The ligated language L(G) = { w ∈ 2 Σ * | S =⇒ * w}.

Matrix Insertion-Deletion Systems

In this section, we describe Matrix insertion-deletion (in short Matrix ins-del) systems. A Matrix ins-del system is a construct Υ = (V, T, A, R) where V is an alphabet, T ⊆ V , A is a finite language over V , R is a finite triples of the form in matrix format

[(u 1 , α 1 /β 1 , v 1 ), . . . , (u n , α n /β n , v n )], where (u k , v k ) ∈ V * × V * , and (β k , α k ) ∈ (V + × {λ}) ∪ ({λ} × V + ), with (u k , α k /β k , v k ) ∈ R Ii ∪ R Dj ∪ R Ii/Dj , for 1 ≤ k ≤ n.
Here R Ii denotes the matrix which consists of only insertion rules, R Dj denotes the matrix which consists of only deletion rules and R Ii/Dj denotes the matrix which consists of both insertion and deletion rules.

Consequently, for x, y ∈ V * we can write x =⇒ y, if y can be obtained from x by using a matrix consisting of insertion or deletion or insertion and deletion rules as follows: In a derivation step the rules in a matrix are applied sequentially one after other in order and no rule is in appearance checking (note that the rules in a matrix are not applied in parallel). The language generated by Υ is defined by

L(Υ) = {w ∈ T * | x =⇒ *
Rχ w, for some x ∈ A}, where χ ∈ {I i , D j , I i /D j } where =⇒ * is the reflexive and transitive closure of the relation =⇒. Note that the string w is collected after applying all the rules in a matrix and also w ∈ T * only. Note that the above mentioned cut, uncut and ligate function are also applicable to Matrix ins-del systems. ♦, # are included in V in Matrix ins-del system.

Modelling Intermolecular structures

In this section, we model some of the intermolecular structures using Matrix insertiondeletion systems.

Lemma 1 The double strand language

L ds = {u♦ū R | u ∈ Σ *
DN A } can be modelled by Matrix ins-del system.

Proof. The language L ds can be be modelled by Matrix ins-del system Υ ds = ({b, b, ♦}, {b, b, ♦}, {♦}, R) where b ∈ {a, t, g, c}, b is complement of b and R is given as: R I1 = [(λ, λ/b, ♦), (♦, λ/ b, λ)] A sample derivation is given as follows:

↓ ♦ ↓ =⇒ RI 1 a ↓ ♦ ↓ t =⇒ RI 1 ag ↓ ♦ ↓ ct =⇒ RI 1 aga ↓ ♦ ↓ tct =⇒ RI 1 agac ↓ ♦ ↓ gtct 2
In the next lemma, we give a cut grammar which generates nick language and we model such cut grammar using Matrix ins-del system. From Fig. 3(b) the nick language can be informally described as

{w 1 ♦w 2 | w 2 = w1
R }, where w 1 ∈ Σ * and w 2 ∈ (Σ ∪ {♦}) * (i.e., w 2 is a string which contains many number of ♦).

Lemma 2

The nick language L nl can be generated by Matrix ins-del system.

Proof. The language L nl can be generated by the cut grammar

G nl = S → bS b | S♦ | ♦ for each b ∈ Σ DN A .
The grammar G nl can be modelled by Matrix ins-del system

Υ nl = ({b, b, †, ♦}, {b, b, ♦}, {b † b, †♦, ♦}, R) where b ∈ {a, t, g, c}, b is complement of b and R is given as: R I1 = [(λ, λ/b, †), ( †, λ/ b, λ)], R I2 = [( †, λ/♦, λ)], R D1 = [(♦, †/λ, λ)].
A sample derivation is given as follows:

a ↓ † ↓ t =⇒ RI 1 at ↓ † ↓ at =⇒ RI 1 atg † ↓ cat =⇒ RI 2 atg ↓ † ↓ ♦cat =⇒ RI 1 atga † ↓ t♦cat =⇒ RI 2 atga † ♦t♦cat =⇒ RD 1 atga ⇓ ♦t♦cat 2
In the next two lemmas we model R-loops and holliday structure as mentioned in Fig. 4 (in Fig 4 . • denotes ♦, * denotes λ and S, A, R represents the non-terminals in their corresponding context-free grammars).

Lemma 3

The hybrid molecule language (with any number of R-loops)

L rl = {u♦vū R | u, v ∈ Σ * DN A
} can be generated by Matrix ins-del system.

Proof. The language L rl can be generated by Matrix ins-del system

Υ rl = ({b, b, † 1 , † 2 , ♦}, {b, b, ♦}, { † 1 ♦ † 2 }, R) where b ∈ {a, t, g, c}, b is complement of b and R is given as: R I1 = [(λ, λ/b, † 1 ), ( † 2 , λ/ b, λ)], R I2 = [(λ, λ/b, † 2 )], R D1 = [(λ, † 1 /λ, λ)], R D2 = [(λ, † 2 /λ, λ)].
A sample derivation is given as follows:

Definition 1:

1. A Matrix ins-del system Υ, is said to be 0-ambiguous, if there exist at least two different axioms, w 1 , w 2 ∈ A, w 1 = w 2 , such that they both derive the same word z, i.e., w 1 =⇒ + z, w 2 =⇒ + z. 2. A Matrix ins-del system Υ, is said to be 1-ambiguous, if there are two different ordered control sequences which derive the same word. 3. A Matrix ins-del system Υ, is said to be 2-ambiguous, if there are two different ordered complete control sequences which derive the same word. 4. A Matrix ins-del system Υ, is said to be 3-ambiguous, if there are two different descriptions which derive the same word.

Interpretation of Ambiguity in Gene Sequences

In this section, our aim is not to show that there exists inherently ambiguous languages, rather our objective is to present a new interpretation of ambiguity in gene sequences.

As an application to the new introduced ambiguity levels, we present some intra and intermolecular structures in gene sequences which exhibits the above introduced new levels of ambiguity for Matrix ins-del systems. Level 0: The Matrix ins-del system is said to be 0-ambiguous if the same string can be derived from two different axioms. Definition 2: A string w over a complementary alphabet Σ is called ideal iff |w| b = |w|b for all b ∈ Σ. A language is ideal iff it contains only ideal strings.

Lemma 7

The ideal language L id generated by Matrix ins-del system exhibits level 0 ambiguity.

Proof. The ideal language L id can be generated by the Matrix ins-del system Υ id = ({b, b}, {b, b}, {λ}, R), where b ∈ {a, t, g, c}, b is complement of b and R is given as R I1 = [(λ, λ/b, λ), (λ, λ/ b, λ)]. Consider the gene sequence tactgagcta in ideal language. This sequence can be generated by the Matrix ins-del system Υ id from two different axioms at and gc such that the same string is obtained at the end of the derivation. The two different derivations which differ by axioms are given as follows:

Derivation 1 : at ↓ =⇒ a ↓ t ↓ gc =⇒ actg ↓ gc ↓ =⇒ ↓ actgagct ↓ =⇒ tactgagcta Derivation 2 : gc ↓ =⇒ ↓ gcta =⇒ ta ↓ g ↓ cta =⇒ ta ↓ tga ↓ cta =⇒ tactgagcta2
Level 1: The Matrix ins-del system is said to be 1-ambiguous if there are two different derivations for the same string which differs by the order of string deleted/inserted.

In the next lemma, we show that level 1 ambiguity exists in both intramolecular structure (stem and loop structure) and intermolecular structure (hybrid molecule structure). Consider the gene sequence aagtt in stem and loop structure. This sequence can be generated in two different ordered control sequences by the Matrix ins-del system Υ sl . Note that the axiom for both sequence is the same. The two sequences are given as follows:

Sequence 1 :

a ↓ † 1 † 3 † ↓ 2 t =⇒ RI 1 aa † ↓ 1 † 3 † 2 tt =⇒ RI 2 aa † 1 g † 3 † 2 tt =⇒ RD 1 aa ⇓ g † ⇓ 3 tt =⇒ RD 2 aag ⇓ tt Sequence 2 : a † ↓ 1 † 3 † 2 t =⇒ RI 2 a ↓ † 1 g † 3 † ↓ 2 t =⇒ RI 1 aa † 1 g † 3 † 2 tt =⇒ RD 2 aa † 1 g ⇓ † 2 tt =⇒ RD 1 aa ⇓ g ⇓ tt 2
In sequence 1, the order of strings used for deletion/insertion is (a, t), g, ( † 1 , † 2 ), † 3 . In sequence 2, the order of strings used for deletion/insertion is g, (a, t), † 3 , ( † 1 , † 2 ). Thus, we obtain two different ordered control sequences which derive the same gene sequence. Therefore, the Matrix ins-del system Υ sl obeys level 1 ambiguity. The Level 1 ambiguity can be pictorially represented as shown in Fig. 5.

Figure 1 .

 1 Figure 1. Intramolecular structures: (a) stem and loop (b) cloverleaf (c) dumbbell

Figure 2 .Figure 3 .

 23 Figure 2. Intramolecular structures: (a) pseudoknot structure (b) attenuator

Lemma 8

 8 The stem and loop structure L sl = {uvū R | u, v ∈ Σ * DN A } represented by Matrix ins-del system obeys level 1 ambiguity. Proof. The stem and loop structure L sl can be generated by the Matrix ins-del system Υ sl = ({b, b, † 1 , † 2 , † 3 }, {b, b}, {λ, b † 1 † 3 † 2 b}, R), where b ∈ {a, t, g, c}, b is complement of b and R is given as: R I1 = [(λ, λ/b, † 1 ), ( † 2 , λ/ b, λ)], R I2 = [(λ, λ/b, † 3 )], R D1 = [(λ, † 1 /λ, λ), (λ, † 2 /λ, λ)], R D2 = [(λ, † 3 /λ, λ)].

Fig. 5 (

 5 a) corresponds to sequence 1 and Fig. 5(b) corresponds to sequence 2.

Figure 5 .

 5 Figure 5. Ambiguity in stem and loop structure

This work was partially supported by the project SR/S3/EECE/054/2010, Department of Science and Technology (DST), New Delhi, India.

Corresponding Author: School of Computing Science and Engineering, VIT University, Vellore 632 014, India.E-mail: klakshma@vit.ac.in

DN A } can be generated by Matrix ins-del system.

Proof. The language L hs (for n=3) can be generated by Matrix ins-del system

Lemma 5 The replication fork language

Proof. The language L rf can be generated by

A sample derivation is given as follows:

The generalized linear hybridization L glh can be modelled by Matrix ins-del system.

Proof. The generalized linear hybridization language L glh can be generated by the grammar

The grammar G glh can be modelled by Matrix ins-del system

A sample derivation is given as follows:

New Levels of Ambiguity

In this section, we formally define various ambiguity levels for Matrix ins-del system based on the components used in the derivation such as axiom, contexts, strings (for deletion/insertion).

Consider the following derivation step in a Matrix ins-del system Υ, δ : w 1 =⇒ w 2 =⇒ . . . ... =⇒ w m , m ≥ 1, such that w 1 ∈ A and the following scenarios can happen (1): w k =⇒ w k+1 can be obtained by using a matrix which consists of only insertion rules (R Ii ) (2): w k =⇒ w k+1 can be obtained by using a matrix which consists of only deletion rules (R Dj ) (3): w k =⇒ w k+1 , such that 1 ≤ k ≤ m can be obtained by using a matrix which consists of both insertion and deletion rules (R Ii/Dj ). The sequence which consists of used axiom, strings α j /β j to be deleted/inserted is called as Control Sequence which is given as follows:

More precisely the control sequence means the order in which the strings are deleted/inserted. The sequence which consists of used axiom, the strings β j /α j to be deleted/inserted and the used contexts (u j , v j ) is called Complete Control Sequence which is given as follows:

More precisely the complete control sequence means the order of the contexts used in deletion/insertion rules. Note that one of β j or α j is empty for all j in the derivation. The position where insertion β /deletion α takes place can be given by the description of δ.

Sequence

In sequence 1, the order of strings used for deletion/insertion is (g, c), (t, a), g, c, † 1 , † 2 . In sequence 2, the order of strings used for deletion/insertion is g, c, (g, c), (t, a), † 2 , † 1 . Thus, we obtain two different ordered control sequences which derive the same gene sequence gt♦gcac. Therefore, the Matrix ins-del system Υ rl exhibits level 1 ambiguity.

Level 2:

The Matrix ins-del system is said to be 2-ambiguous if there are two different derivations for the same string which differs by the order of contexts used for deletion/insertion.

Lemma 9

The holliday structure

DN A } represented by Matrix ins-del system obeys level 2 ambiguity.

Proof. Consider the following string t♦ac♦gg♦c in holliday structure. This string can be generated in two different ordered complete control sequences by the Matrix ins-del system Υ hs . The two sequences are given below:

In sequence 1, the order of contexts used is

In sequence 2, the order of contexts used is

. Thus, we obtain two different ordered complete control sequences which derive the same gene sequence t♦ac♦gg♦c. Therefore, the Matrix ins-del system Υ hs obeys level 2 ambiguity.

Level 3: The Matrix ins-del systems is said to be 3-ambiguous if there are two different descriptions for the same string which differs by the position where the string is deleted/inserted. Definition 3: A string w over a complementary alphabet Σ is called orthodox iff it is (i) the empty string λ, or (2) the result of inserting two adjacent complementary element b b, for some b ∈ Σ, anywhere in an orthodox string. A language is orthodox iff it contains only orthodox strings.

Lemma 10

The orthodox language L od generated by Matrix ins-del system obeys level 3 ambiguity.

Proof. The orthodox language L od can be generated by the Matrix ins-del system Υ od = ({b, b}, {b, b}, {λ}, R), where b ∈ {a, t, g, c}, b is complement of b and R is given as

Consider the string tagcgcat in orthodox language. This string can be derived in two different descriptions by Υ od which are given as follows:

Note that the axiom, order of insertion of strings, order of contexts (here (λ, λ)) all are same in both derivations, but the position of insertion is different in each derivation. Therefore, the system Υ od is 3-ambiguous. Note: Consider the orthodox string gcgcgcgc. This string can be derived from axiom λ by two different descriptions which are given as follows:

In one derivation the string gc is used one time and cg is used three times. In another derivation the string gc is used four times. The above string obeys level 1 ambiguity but the intermediate gene sequences are same.

Conclusion

In this paper, we discussed the Matrix insertion-deletion grammar systems and using the system we have modelled some intermolecular structures like double strand languages, nick languages, hybrid molecules (with R-loops), holliday structure, replication fork, linear hybridization (ligated) languages. We have formally defined various levels of i (i = 0, 1, 2, 3)-ambiguity for Matrix insertion-deletion systems. We have given the application for the introduced levels of ambiguity with an interpretation in gene sequences. We witnessed that the many gene sequences in intra and intermolecular structures like ideal, stem and loop, hybrid molecule, holliday structure, orthodox has a relevance with the introduced levels of ambiguity. Since, in this paper we have shown that ambiguity is possible in gene sequences this research will throw some ideas on how gene sequences in DNA, RNA, protein molecules can be predicted and processed.


Jan Kehrmann

Martin Kaase

Florian Szabados

Soeren G Gatermann

Jan Buer

Peter-Michael Rath

MD Joerg Steinmann
email: joerg.steinmann@uk-essen.de

Vancomycin MIC creep in MRSA blood culture isolates from Germany: a regional problem?

Keywords: vancomycin MIC creep, MRSA, Etest

Purpose: The aim of this study was to assess the vancomycin MIC distribution for MRSA blood culture isolates over a period of six years in Germany.

Methods: The study examined 287 MRSA isolates from blood cultures collected at several hospitals in two German cities between 2004 and 2009. The vancomycin MIC was determined by Etest. Genotypic features of the MRSA strains with vancomycin MIC ≥1 mg/L were determined by semiautomated repetitive-sequencebased polymerase chain reaction.

Results: The range of vancomycin MIC as determined by Etest was 0.25 to 2.0 mg/L. The geometric mean MIC increased by 1.34-fold in city A over the study period (p<0.05), but there was no meaningful change in city B (a 1.09-fold increase, p>0.05). Furthermore, in city A a shift in vancomycin MICs occurred as an increase in the percentage of isolates with MIC ≥1 mg/L from period one (2004)(2005)(2006) to period two (2007)(2008)(2009) (p<0.0001). Typing results showed that in city A a single clone was predominant (55% of the creep isolates).

Conclusions: In this study, the creep phenomenon seems to be a regional problem. We suggest that all hospitals should monitor their local status of elevated vancomycin MICs in invasive MRSA isolates.

Introduction

Staphylococcus aureus is among the most common pathogens causing hospital-and communityacquired infections and is one of the leading causes of sepsis worldwide. In Europe, S. aureus is the second most common causative organism for bacteraemia [START_REF] Biedenbach | Occurrence and antimicrobial resistance pattern comparisons among bloodstream infection isolates from the SENTRY Antimicrobial Surveillance Program (1997-2002)[END_REF]. Adequate and early antibiotic therapy is a crucial factor in the prognosis of sepsis [START_REF] Soriano | Pathogenic significance of methicillin resistance for patients with Staphylococcus aureus bacteremia[END_REF][START_REF] Ibrahim | The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the ICU setting[END_REF][START_REF] Kumar | Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock[END_REF]. The antibiotic treatment of S. aureus infections is complicated by the increasing prevalence of methicillin-resistant S. aureus (MRSA) [START_REF] Biedenbach | Occurrence and antimicrobial resistance pattern comparisons among bloodstream infection isolates from the SENTRY Antimicrobial Surveillance Program (1997-2002)[END_REF][START_REF] Tiemersma | Staphylococcus aureus bacteremia, Europe[END_REF]. Infections caused by MRSA are associated with a higher mortality rate than are those associated with methicillin-susceptible S. aureus (MSSA) [START_REF] Cosgrove | Comparison of mortality associated with methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteremia: a meta-analysis[END_REF][START_REF] Whitby | Risk of death from methicillin-resistant Staphylococcus aureus bacteraemia: a meta-analysis[END_REF], and they require a longer hospital stay and higher costs. In the United States, nearly half of all S. aureus isolates from patients are MRSA [START_REF] Styers | Laboratory-based surveillance of current antimicrobial resistance patterns and trends among Staphylococcus aureus: 2005 status in the United States[END_REF], whereas in Germany approximately 20% of such isolates are MRSA [START_REF] Kresken | Resistenzsituation bei klinisch wichtigen Infektionserregern gegenüberAntibiotika in Deutschland und im mitteleuropäischen Raum[END_REF].

The first antibiotic agent and today still the gold standard for the treatment of invasive MRSA infections is the glycopeptide vancomycin. The first isolate with reduced vancomycin susceptibility (minimum inhibitory concentration [MIC], 8 mg/L) was reported in Japan in 1997 [START_REF] Hiramatsu | Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility[END_REF]. Only a few strains of vancomycin-resistant S. aureus (VRSA) and vancomycin-intermediate S. aureus (VISA) have been reported to exhibit resistance to vancomycin [START_REF] Tenover | Characterization of staphylococci with reduced susceptibilities to vancomycin and other glycopeptides[END_REF][START_REF] Van Griethuysen | High percentage of methicillin-resistant Staphylococcus aureus isolates with reduced susceptibility to glycopeptides in The Netherlands[END_REF]. Nevertheless, the number of patients for whom vancomycin therapy fails is increasing, even though their S. aureus strains have been found to be susceptible to vancomycin in vitro (MIC ≤2 mg/L) [START_REF] Moise | Vancomycin treatment failures in Staphylococcus aureus lower respiratory tract infections[END_REF][START_REF] Soriano | Influence of vancomycin minimum inhibitory concentration on the treatment of methicillinresistant Staphylococcus aureus bacteremia[END_REF][START_REF] Howden | Treatment outcomes for serious infections caused by methicillin-resistant Staphylococcus aureus with reduced vancomycin susceptibility[END_REF]. Having analyzed clinical and microbiological data, several groups of researchers have concluded that increasing vancomycin MICs in MRSA are associated with a substantial risk of treatment failure and with increased mortality rates [START_REF] Soriano | Influence of vancomycin minimum inhibitory concentration on the treatment of methicillinresistant Staphylococcus aureus bacteremia[END_REF][START_REF] Howden | Treatment outcomes for serious infections caused by methicillin-resistant Staphylococcus aureus with reduced vancomycin susceptibility[END_REF][START_REF] Charles | Clinical features associated with bacteremia due to heterogeneous vancomycin-intermediate Staphylococcus aureus[END_REF][START_REF] Wang | Nosocomial methicillin-resistant Staphylococcus aureus (MRSA) bacteremia in Taiwan: mortality analyses and the impact of vancomycin, MIC = 2 mg/L, by the broth microdilution method[END_REF].

Several studies have demonstrated that the mean vancomycin MIC in MRSA has increased in recent years [START_REF] Steinkraus | Vancomycin MIC creep in non-vancomycin-intermediate Staphylococcus aureus (VISA), vancomycin-susceptible clinical methicillin-resistant S. aureus (MRSA) blood isolates from 2001-05[END_REF][START_REF] Wang | Increased vancomycin MICs for Staphylococcus aureus clinical isolates from a university hospital during a 5-year period[END_REF][START_REF] Rybak | Characterization of vancomycin-heteroresistant Staphylococcus aureus from the metropolitan area of Detroit, Michigan, over a 22-year period (1986 to 2007)[END_REF][START_REF] Ho | Vancomycin MIC creep in MRSA isolates from 1997 to 2008 in a healthcare region in Hong Kong[END_REF], but other studies have not confirmed this finding [START_REF] Sader | Evaluation of vancomycin and daptomycin potency trends (MIC creep) against methicillin-resistant Staphylococcus aureus isolates collected in nine U.S. medical centers from 2002 to 2006[END_REF][START_REF] Alós | Vancomycin MICs did not creep in Staphylococcus aureus isolates from 2002 to 2006 in a setting with low vancomycin usage[END_REF]. The observation that vancomycin MICs are increasing within the susceptible range of MRSA is called the creep phenomenon.

As a result of published reports showing that vancomycin therapy fails for patients who have S. aureus infections with a MIC ≥4 mg/L, the Clinical and Laboratory Standards Institute (CLSI) reduced the pre-2006 vancomycin breakpoints from ≤4 mg/L to ≤2 mg/L for susceptible S. aureus and from ≥32 mg/L to ≥16 mg/L for resistant S. aureus [START_REF] Tenover | The rationale for revising the Clinical and Laboratory Standards Institute vancomycin minimal inhibitory concentration interpretive criteria for Staphylococcus aureus[END_REF]. Some experts believe that these breakpoints are still too high and that a further reduction is necessary to prevent a higher risk of therapy failure for MRSA creep strains and to anticipate future MIC creep trends [START_REF] Gould | The problem with glycopeptides[END_REF].

Only a few studies have examined the creep phenomenon in Europe, and to the best of our knowledge no studies have examined the creep phenomenon in Germany. Thus, the aim of this study was to analyze the vancomycin MIC distribution in MRSA blood culture obtained over a period of six years in western Germany.

Material and methods

Isolates

In this study we analyzed MRSA blood culture isolates obtained from patients in several hospitals in two cities of the Ruhr region of North Rhine Westphalia between January 2004 and December 2009. Only one isolate per patient was tested. For those patients with more than one isolate, only the first isolate was analyzed.

In all, 133 isolates were collected from a tertiary-care hospital in Essen (city A) and 154 from four secondarycare hospitals in Bochum (city B). Germany) or MicroScan® Walkaway (Siemens, Erlangen, Germany). These same commercial systems were also used for susceptibility testing of oxacillin. Individual isolates were then frozen at -80°C until MIC testing was performed.

Vancomycin MIC analysis

Vancomycin MIC testing was performed by the Etest method, according to the manufacturer´s guidelines. A suspension in saline to the 0.5 McFarland turbidity standard was plated on Mueller-Hinton agar, and Etest strips were applied (AB BIODISK, Solna, Sweden, now distributed by bioMérieux). The plates were incubated at 35°C for 24 hours. MIC was interpreted as the zone of inhibition corresponding to a concentration gradient on the Etest strips, according to the manufacturer's guidelines. Quality control was performed with the CLSI-recommended reference strain (ATCC 29213).

Data analysis and statistical methods

The MIC 50 , MIC 90 , MIC range, and geometric mean MIC were evaluated. All calculations were performed for each year, using only those isolates collected in that calendar year. Non-parametric methods (Spearman correlation and chi-square test) were used to asses MIC trends over 6 years. Statistical significance was defined a priori as p<0.05. Statistical analyses were performed with GraphPad Prism, version 5.02 (GraphPad Software, Inc, San Diego, CA, USA).

Genotyping

Isolates were typed with the semiautomated repetitive-sequence-based polymerase chain reaction (PCR) DiversiLab system (bioMérieux), according to the manufacturer's instructions. Genomic DNA isolation of MRSA isolates was performed with the Mo Bio Ultra Clean preparation kit (bioMérieux). After DNA extraction, the concentration was quantified with a nanodrop1000 spectrometer (Peqlab, Erlangen, Germany) and software

Results

This study analyzed a total of 287 blood culture isolates collected from 287 patients in two German cities over a period of six years between 2004 and 2009. All MRSA isolates from both cities were tested as vancomycin-susceptible in vitro. The vancomycin MIC ranged from 0.25 to 2.0 mg/L (Table 1A and1B). For the isolates from city A, the geometric mean vancomycin MIC increased by 1.34-fold during the study period: from 0.59 mg/L in 2004 to 0.79 mg/L in 2009 (p=0.042) (Figure 1A). In contrast, the geometric mean vancomycin MIC for the isolates from city B exhibited only a slight increase (1.09-fold; not statistically significant) during the study period: from 0.47 mg/L in 2004 to 0.51 mg/L in 2009 (p>0.05) (Figure 1B). For the isolates from city A, the percentile markers for MIC 50 increased from 0.5 mg/L to 0.75 mg/L, and those for MIC 90 increased from 0.75 mg/L to 1.0 mg/L. For city B, in contrast, the percentile markers for MIC 50 (0.5 mg/L) and MIC 90 (0.75 mg/L) remained unchanged during the study period. The increase in the mean vancomycin MIC in city A was primarily due to a significant increase in the percentage of isolates with a MIC ≥1 mg/L from period one (2004)(2005)(2006) to period two (2007-2009) (p<0.0001). The fraction of these isolates with a MIC ≥1 mg/L increased from 6.7% in 2004 to 20.8% in 2007 and 35.7% in 2009, whereas only two of the isolates from city B had a vancomycin MIC ≥1 mg/L.

All MRSA isolates with a vancomycin MIC ≥1 mg/L were typed with the semiautomatic sequencebased PCR DiversiLab system. This testing included 18 MRSA isolates from city A and two isolates from city B. Eleven of these 20 isolates, all from city A, exhibited a similarity of 96% or higher, a finding indicating a clonal evolution in these isolates; this evolution involved 55% of all isolates with a MIC ≥1 mg/L (Figure 2). The first of these isolates was obtained from a patient treated in 2004; the last, from a patient treated in 2009.

The isolates were obtained from patients hospitalized in different clinical departments. The two isolates from city B did not belong to this clonal cluster.

Discussion

This study analyzed the vancomycin MIC of 287 MRSA blood culture isolates collected in several hospitals in two German cities between 2004 and 2009. The vancomycin MICs for MRSA blood culture isolates from city A increased significantly over the study period, but there was no significant increase in vancomycin MICs for MRSA blood culture isolates from city B. To the best of our knowledge, this study is the first to analyze the existence of a vancomycin creep phenomenon for MRSA in Germany. Typing of the isolates with a vancomycin MIC ≥1 mg/L showed that a single clone was predominant and was responsible for 55% of these isolates.

The interest in analyzing vancomycin MICs is not only originated from concern that a further creep could shift MICs into the non-susceptible zone. There have been several reports of treatment failure against vancomycin-susceptible isolates [START_REF] Soriano | Influence of vancomycin minimum inhibitory concentration on the treatment of methicillinresistant Staphylococcus aureus bacteremia[END_REF][START_REF] Howden | Treatment outcomes for serious infections caused by methicillin-resistant Staphylococcus aureus with reduced vancomycin susceptibility[END_REF][START_REF] Charles | Clinical features associated with bacteremia due to heterogeneous vancomycin-intermediate Staphylococcus aureus[END_REF][START_REF] Lodise | Relationship between vancomycin MIC and failure among patients with methicillin-resistant Staphylococcus aureus bacteremia treated with vancomycin[END_REF][START_REF] Sakoulas | Relationship of MIC and bactericidal activity to efficacy of vancomycin for treatment of methicillinresistant Staphylococcus aureus bacteremia[END_REF][START_REF] Jones | Microbiological features of vancomycin in the 21st century: minimum inhibitory concentration creep, bactericidal/static activity, and applied breakpoints to predict clinical outcomes or detect resistant strains[END_REF]. Patients with a MRSA bloodstream infection with an elevated vancomycin MIC (≥1.5 mg/L) were reported to have a longer duration of bacteraemia, a higher probability of recurrent bacteraemia within 60 days after vancomycin discontinuation, and a longer hospital stay [START_REF] Lodise | Relationship between vancomycin MIC and failure among patients with methicillin-resistant Staphylococcus aureus bacteremia treated with vancomycin[END_REF]. The likelihood of treatment success was significantly lower for patients with MRSA isolates with a vancomycin MIC of 1 to 2 mg/L than for patients with a vancomycin MIC ≤0.5 mg/L [START_REF] Sakoulas | Relationship of MIC and bactericidal activity to efficacy of vancomycin for treatment of methicillinresistant Staphylococcus aureus bacteremia[END_REF]. Treatment with vancomycin was successful for 55.6% of the patients with a vancomycin MIC ≤0.5 mg/L but for only 9.5% of those with a vancomycin MIC of 1 to 2 mg/L [START_REF] Sakoulas | Relationship of MIC and bactericidal activity to efficacy of vancomycin for treatment of methicillinresistant Staphylococcus aureus bacteremia[END_REF].

The phenomenon of an increasing vancomycin MIC for MRSA has been demonstrated by several single-centre studies in the United States [START_REF] Steinkraus | Vancomycin MIC creep in non-vancomycin-intermediate Staphylococcus aureus (VISA), vancomycin-susceptible clinical methicillin-resistant S. aureus (MRSA) blood isolates from 2001-05[END_REF][START_REF] Wang | Increased vancomycin MICs for Staphylococcus aureus clinical isolates from a university hospital during a 5-year period[END_REF][START_REF] Rybak | Characterization of vancomycin-heteroresistant Staphylococcus aureus from the metropolitan area of Detroit, Michigan, over a 22-year period (1986 to 2007)[END_REF] and by a study from the health care region of Hong Kong [START_REF] Ho | Vancomycin MIC creep in MRSA isolates from 1997 to 2008 in a healthcare region in Hong Kong[END_REF].

An examination of the extent of the vancomycin creep found a more than three-fold increase in the proportion of strains with MIC = 1 mg/L [START_REF] Steinkraus | Vancomycin MIC creep in non-vancomycin-intermediate Staphylococcus aureus (VISA), vancomycin-susceptible clinical methicillin-resistant S. aureus (MRSA) blood isolates from 2001-05[END_REF][START_REF] Wang | Increased vancomycin MICs for Staphylococcus aureus clinical isolates from a university hospital during a 5-year period[END_REF]. During the same period, the proportion of strains with MIC ≤0.5 mg/L showed a decrease of a similar proportion [START_REF] Wang | Increased vancomycin MICs for Staphylococcus aureus clinical isolates from a university hospital during a 5-year period[END_REF]. Wang et al. found only a small increase in the proportion of strains with MIC ≥2.0 mg/L [START_REF] Wang | Increased vancomycin MICs for Staphylococcus aureus clinical isolates from a university hospital during a 5-year period[END_REF], and Steinkraus et al. found no non-susceptible strains [START_REF] Steinkraus | Vancomycin MIC creep in non-vancomycin-intermediate Staphylococcus aureus (VISA), vancomycin-susceptible clinical methicillin-resistant S. aureus (MRSA) blood isolates from 2001-05[END_REF]. Our findings regarding the isolates from city A agree with these results: we found an increasing proportion of strains with vancomycin MICs ≥1 mg/L and a decreasing proportion of strains with MICs ≤0.5 mg/L; in addition, we found no intermediate strains (MIC >2 mg/L and ≤4 mg/L). In contrast to the findings of reported studies, we also found increasing percentile markers of MIC 50 and MIC 90 for the isolates from city A.

The isolates from city B did not show a relevant increase in vancomycin mean MIC or in MIC 50 or MIC 90 . These results are consistent with those of a multi-centre study of nine US medical centres that found no increase in vancomycin MIC for all sites between 1999 and 2006. Vancomycin MIC increased at three sites but decreased at three other sites [START_REF] Sader | Evaluation of vancomycin and daptomycin potency trends (MIC creep) against methicillin-resistant Staphylococcus aureus isolates collected in nine U.S. medical centers from 2002 to 2006[END_REF]. A survey from Spain found no MIC creep in MSSA or MRSA isolates from 2002 to 2006 [START_REF] Alós | Vancomycin MICs did not creep in Staphylococcus aureus isolates from 2002 to 2006 in a setting with low vancomycin usage[END_REF]. Also, the SENTRY Antimicrobial Surveillance Program database for the years 1998 to 2003, which included 35,458 MSSA and MRSA isolates, found no change in vancomycin MIC over the study period [START_REF] Jones | Microbiological features of vancomycin in the 21st century: minimum inhibitory concentration creep, bactericidal/static activity, and applied breakpoints to predict clinical outcomes or detect resistant strains[END_REF].

However, the question of why a creep phenomenon has been detected at some centres but not all has not yet been sufficiently answered. Because of the heterogeneity of the studies performed to date, a direct comparison is difficult. The studies differ in the periods analyzed, the methods used for susceptibility testing (Etest vs. broth dilution), and the type of isolates analyzed (MRSA vs. MSSA and blood culture vs. all clinical isolates).

The two methods primarily used in previously published to analyze the vancomycin MIC in MSSA and MRSA, Etest and broth dilution, yielded different results. The findings generated by Etest were a single two-fold dilution higher than those generated by CLSI broth dilution [START_REF] Prakash | Vancomycin MICs for methicillin-resistant Staphylococcus aureus isolates differ based upon the susceptibility test method used[END_REF]. The Etest method is more sensitive in detecting small vancomycin MIC changes than the broth dilution method, provided that the traditional two-fold dilution MIC testing is performed. The studies that did not find a vancomycin MIC creep used primarily the broth dilution method, whereas most studies that did find a vancomycin MIC creep used the Etest method [START_REF] Steinkraus | Vancomycin MIC creep in non-vancomycin-intermediate Staphylococcus aureus (VISA), vancomycin-susceptible clinical methicillin-resistant S. aureus (MRSA) blood isolates from 2001-05[END_REF][START_REF] Wang | Increased vancomycin MICs for Staphylococcus aureus clinical isolates from a university hospital during a 5-year period[END_REF][START_REF] Rybak | Characterization of vancomycin-heteroresistant Staphylococcus aureus from the metropolitan area of Detroit, Michigan, over a 22-year period (1986 to 2007)[END_REF][START_REF] Ho | Vancomycin MIC creep in MRSA isolates from 1997 to 2008 in a healthcare region in Hong Kong[END_REF]. The Etest method appears to be more reliable than the broth dilution and semiautomated systems in correlating treatment response and an elevated MIC >1 mg/L [START_REF] Hsu | Comparison of method-specific vancomycin minimum inhibitory concentration values and their predictability for treatment outcome of meticillin-resistant Staphylococcus aureus (MRSA) infections[END_REF].

Unfortunately, most studies describing a vancomycin MIC creep phenomenon did not analyze the strains with a high vancomycin MIC by molecular typing methods to determine whether the creep was due to a dissemination of clonal MRSA clusters. Recently, Ho et al. reported their finding of an increased percentage of clones that lead to a vancomycin creep among MRSA blood culture isolates in Hong Kong [START_REF] Ho | Vancomycin MIC creep in MRSA isolates from 1997 to 2008 in a healthcare region in Hong Kong[END_REF]. We used molecular typing by DiversiLab and found that a single predominant clone accounted for 55% of the isolates with a vancomycin MIC ≥1 mg/L. Interestingly, a recent molecular-epidemiological study of the geographic distribution of invasive MSSA and MRSA isolates in Europe showed a restriction on only a few predominant MRSA clones that clustered regionally [START_REF] Grundmann | Geographic distribution of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis[END_REF]. In that study, multilocus sequence typing demonstrated that, in the analyzed regions in Germany, two MRSA spa types were responsible for more than 60 % of invasive isolates.

Because existing findings indicate that the probability of therapy failure is higher for invasive MRSA infections caused by strains with an increased vancomycin MIC, and because a creep phenomenon appears to exist in some regions, we suggest that every medical centre should examine the local situation of vancomycin MICs in MRSA isolates from blood cultures by Etest. Furthermore, if a vancomycin MIC creep is found in MRSA strains in the patient population of a medical centre, the centre's decision-makers should include in their discussions about the appropriate therapy the main risk factors associated with the patients' acquisition of MRSA with elevated vancomycin MICs. Those factors are exposure to vancomycin during the past month and the collection of the isolate while the patient is in an ICU [START_REF] Lodise | Predictors of high vancomycin MIC values among patients with methicillin-resistant Staphylococcus aureus bacteraemia[END_REF]. Increasing the dosage of vancomycin given to MRSA patients with elevated vancomycin MICs does not seem to improve outcome; instead, it may impair renal function, particularly when patients are concurrently taking a nephrotoxic medication [START_REF] Hidayat | High-dose vancomycin therapy for methicillin-resistant Staphylococcus aureus infections: efficacy and toxicity[END_REF]. For invasive MRSA infections with strains that have an elevated vancomycin MIC, physicians might consider an alternative therapy, such as daptomycin or linezolid, both of which have been reported to exhibit high in vitro activity [START_REF] Laplante | Impact of high-inoculum Staphylococcus aureus on the activities of nafcillin, vancomycin, linezolid, and daptomycin, alone and in combination with gentamicin, in an in vitro pharmacodynamic model[END_REF][START_REF] Sakoulas | Daptomycin in the treatment of bacteremia[END_REF] and to be effective in treating invasive MRSA infections in clinical trials [START_REF] Sakoulas | Daptomycin in the treatment of bacteremia[END_REF][START_REF] Wunderink | Linezolid vs vancomycin: analysis of two double-blind studies of patients with methicillin-resistant Staphylococcus aureus nosocomial pneumonia[END_REF][START_REF] Fowler Vg | aureus Endocarditis and Bacteremia Study Group (2006) Daptomycin versus standard therapy for bacteremia and endocarditis caused by Staphylococcus aureus[END_REF].

This study is limited by the fact that it examined MRSA blood culture isolates from only two cities in the Ruhr area; therefore, the findings may not be representative of all of Germany. Another limitation is the lack of clinical information about the outcome of the patients. Nevertheless, this is not a clinical trial but is rather an epidemiological study analyzing the vancomycin MIC.

In summary, this study compared the vancomycin MIC distribution in two German cities. A vancomycin MIC creep phenomenon was detected at one site on the basis of a significant increase in the number of isolates with a MIC of 1 mg/L or higher. Typing showed that a single clone was predominant for 55% of these isolates. The vancomycin MIC creep phenomenon seems to be a regional problem and we suggest that all hospitals should monitor their local status of elevated vancomycin MICs in invasive MRSA isolates. More studies will be necessary to verify our observation and to confirm the situation in the entire country of Germany.

version 3 . 7 . 1 .

 371 Genomic DNA concentrations were adjusted to approximately 25 ng/µL as recommended by the manufacturer. The bacterial DNA was amplified with the DiversiLab Staphylococcus aureus kit (bioMérieux) for DNA fingerprinting. In brief, 25 ng of DNA, 2.0 µL of the rep-PCR primer mixture provided with the kits, 0.5 µL of AmpliTaq® (Applied Biosystems, Foster City, CA, USA), 2.5 µL of 10x PCR buffer (Applied Biosystems), and 18 µL of the kit-supplied rep-PCR master mix (MM1) were added, for a total of 25 µL per reaction mixture. Thermal cycling parameters were as follows: initial denaturation at 94ºC for 2 min; 35 cycles of denaturation at 94ºC for 30 s; annealing at 45ºC for 30 s; extension at 70ºC for 90 s; and final extension at 70ºC for 3 min. Separation and detection of the amplified fragments of various sizes and fluorescence intensities were performed by using the micro-fluid chips of the DiversiLab system and a model B 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The results were illustrated with the Web-based DiversiLab software version v.r.3.3.40, which uses the Pearson correlation coefficient and the unweighted-pair group method with arithmetic mean to analyze and determine the genetic similarity of the tested samples. Dendrogram and virtual gel images were automatically generated by the software. Cluster analysis, based on peak height and on the presence and absence of the peaks, was performed with DiversiLab. Percentage similarity for genotyping and discrimination of MRSA isolates with vancomycin MIC ≥1 mg/L was set at a cut-off of 96%.

Figure 1A .Figure 1B .Figure 2 .

 1A1B2 Figure 1A. Vancomycin minimum inhibitory concentration (MIC) trends from city A, 2004-2009

  The two cities are separated by approximately 15 km. Of the 287 isolates, 44

	were collected in 2004, 49 in 2005, 59 in 2006, 54 in 2007, 54 in 2008, and 27 in 2009. All isolates were
	identified as S. aureus by conventional standardized laboratory methods (e.g., growth conditions, morphological
	criteria, pigment production) and by one of two semiautomated systems: Vitek® 2 (bioMérieux, Nürtingen,

Table 1B .

 1B Methicillin-resistant Staphylococcus aureus (MRSA) vancomycin MIC statistics from city B

			Isolates from city B
	Year	Number of	Geometric mean	MIC range	MIC 50	MIC 90 MIC ≥1 mg/L
		strains	[mg/L]	[mg/L]	[mg/L]		[%]
	2004	29	0.47	0.25-0.75	0.5	0.75	0
	2005	25	0.44	0.25-0.75	0.38	0.75	0
	2006	29	0.48	0.25-1.0	0.5	0.75	3.45
	2007	30	0.46	0.25-0.75	0.5	0.5	0
	2008	28	0.46	0.38-0.75	0.5	0.5	0
	2009	13	0.51	0.38-1.0	0.5	0.75	7.69
	MIC: Minimum inhibitory concentration

Acknowledgment

We thank Melanie Wilmes and Dirk Schmidt for their excellent technical assistance.


Eghbali Jan Cosyn
email: jan.cosyn@ugent.be

Aryan Eghbali

Hugo De Bruyn

Kristiaan Collys

Roberto Cleymaet

De Bruyn

Cleymaet, R De Rouck

Tim De Rouck

CONFLICT OF INTERESTS AND SOURCE OF FUNDING

Keywords: dental implant, single tooth, immediate, maxilla, pink esthetic score Dental implant, single tooth, immediate, maxilla, pink esthetic score, white esthetic score

Introduction

Single implant treatment in the anterior maxilla is considered highly predictable and successful, at least in terms of implant survival and hard tissue remodelling following conventional implant surgery [START_REF] Creugers | A systematic review of single-tooth restorations supported by implants[END_REF][START_REF] Berglundh | A systematic review of the incidence of biological and technical complications in implant dentistry reported in prospective longitudinal studies of at least 5 years[END_REF][START_REF] Den Hartog | Treatment outcome of immediate, early and conventional single-tooth implants in the aesthetic zone: a systematic review to survival, bone level, soft-tissue, aesthetics and patient satisfaction[END_REF][START_REF] Jung | A systematic review of the 5-year survival and complication rate of implant-supported single crowns[END_REF]. Hitherto, aesthetic aspects of therapy have only been sporadically reported even though these are more and more becoming the key for success in daily practice.

The latter may be a reflection of an evolving society with more demanding patients expecting a restoration to be an exact copy of the original tooth and finalized within the shortest possible time span. Consequently, implant protocols have been reassessed ultimately resulting in immediate implant placement and restoration. Albeit this strategy offers obvious advantages such as time gain, immediate aesthetics and comfort, immediate implantation may not avert post-extraction remodelling [START_REF] Botticelli | Hard-tissue alterations following immediate implant placement in extraction sites[END_REF][START_REF] Araújo | Ridge alterations following implant placement in fresh extraction sockets: an experimental study in the dog[END_REF]). As such, immediate single implant treatment may be a risky procedure in terms of soft tissue stability especially when patients are improperly selected and surgery is performed by inexperienced clinicians (De Rouck et al. 2008b). To reduce the risk for advanced midfacial soft tissue recession a number of prerequisites have been described (De Rouck et al. 2008b). At least in the short term, immediate implant placement has shown to be predictable under these conditions (Kan et al. 2003a, De Rouck et al. 2008a). The purpose of the present study was to document the overall outcome of immediate single implant treatment in the anterior maxilla in the mid-long term focusing on soft tissue dynamics and aesthetic aspects. The research hypothesis was that this treatment concept would demonstrate a low risk for advanced midfacial recession after 3 years of function.

Material and Methods

Patient selection

This prospective study was based on data from patients who had been treated for immediate single-tooth implants at the Dental Clinic of the Free University in Brussels (VUB). The 1-year results were previously published by De Rouck and co-workers (2008a). This paper addressed the outcome of the same study sample after 3 years of function focusing on soft tissue dynamics between the 1-and 3-year reassessment and aesthetic aspects. Patients were selected during a screening visit on the basis of inclusion and exclusion criteria.

Inclusion criteria were as follows:

1. At least 18 years old.

2. Good oral hygiene defined as full-mouth plaque score ≤ 25 % (O' Leary et al. 1972).

3. Presence of a single failing tooth in the anterior maxilla (15-25) with both neighbouring teeth present. The study was conducted in accordance with the Helsinki declaration of 1975 as revised in 2000.

Surgical procedure

Following screening, comprehensive clinical and radiographic examination was performed by two experienced clinicians (JC/TDR). All patients consented to the planned treatment strategy. The surgical procedure can be found in detail in a previous paper (De Rouck et al. 2008a). Briefly, patients were advised to start antibiotic (Amoxicillin 500 mg) and analgesic therapy (Ibuprofen 600 mg) one hour pre-operatively and oral disinfection (Corsodyl ® , GlaxoSmithKline, Genval, Belgium) was done just prior to surgery. Teeth scheduled for immediate replacement were removed as atraumatically as possible using periotomes after minimal mucoperiosteal flap elevation including the papillae of both adjacent teeth. Immediate implant placement (Nobelreplace tapered TiUnite ® , Nobel Biocare, Göteborg, Sweden) was performed if the buccal bone crest was intact. Special attention was paid to a correct selection and three-dimensional positioning of the implant as described by [START_REF] Buser | Optimizing esthetics for implant restorations in the anterior maxilla: anatomic and surgical considerations[END_REF]. Following confirmation of the primary stability using a Torque Controller (Nobel Biocare, Göteborg, Sweden), implant impression was made. Deproteinized bovine bone particles (Bio-Oss ® 0.25 -1 mm, Geistlich Biomaterials, Wolhusen, Switzerland) soaked in blood were inserted to fill the void between the implant and the alveolus. Finally, an appropriate healing abutment was installed and the wound was closed by means of single sutures (Vicryl ® 5/0, Johnson & Johnson, St-Stevens-Woluwe, Belgium). All surgical procedures were performed by one and the same experienced implant surgeon (JC). For details on the restorative and technical procedures, we wish to refer to a previous paper (De Rouck et al. 2008a). Briefly, an individualized screw-retained provisional crown was fabricated in the dental laboratory using the implant impression taken at the time of surgery. An engaging titanium temporary abutment (Nobel Biocare, Göteborg, Sweden) served as a carrier for an appropriate hollowed denture tooth. Approximately 3 h following implant installation the temporary crown was placed and tightened by one and the same experienced prosthodontist (TDR) at 15 Ncm onto the fixture. The provisional restoration was adjusted to clear centric and eccentric contacts in order to avoid full functional load.

After 6 months, the provisional restoration was replaced by a permanent cemented restoration. A standard aesthetic titanium abutment (Esthetic Abutment, Nobel Biocare, Göteborg, Sweden) was used to connect the permanent metal-ceramic restoration. Cementation was performed using temporary cement (Temp-Bond NE ® , Kerr, Scafati, Italy). All prosthetic procedures were conducted by one and the same experienced prosthodontist (TDR) and all permanent restorations were fabricated in one and the same dental laboratory (Dental Art, Zottegem, Belgium).

Implant survival and complications

As performed during the first year of function, patients were evaluated for implant survival and complications after 3 years. A distinction was made between biologic and technical complications.

Hard tissue parameters

As performed during the first year of function, a peri-apical radiograph using the long-cone paralleling technique was taken after 3 years. Each X-ray holder (XCP Bite Block ® , Dentsply Rinn, Elgin, IL, USA) had been individualized with an occlusal jig (Futar D Fast ® , Kettenbach Dental, Eschenburg, Germany) in order to standardize the procedure. Changes in marginal bone levels at the mesial and distal aspect of the implant were determined following digitalization (SprintScan 35 Plus ® , Polaroid, Cambridge, MA, USA) and by using the appropriate software (Vixwin 2000 v1.11 ® , Dentsply Gendex, Lake Zurich, Switserland). Details can be found in a preceding paper (De Rouck et al. 2008a).

Soft tissue parameters

As performed during the first year of function, the clinical condition of the implant restoration was evaluated after 3 years by means of the following parameters: 1. Plaque score. A dichotomous score was given (0= no visible plaque at the soft tissue margin; 1= visible plaque at the soft tissue margin) at four sites per implant (mesial, midfacial, distal, palatal).

2. Probing depth. It was measured to nearest 0.5 mm at four sites per implant (mesial, midfacial, distal, palatal) using a manual probe (CP 15 UNC, Hu-Friedy ® , Chicago, USA).

Bleeding on probing.

A dichotomous score was given (0= no bleeding; 1= bleeding) at four sites per implant (mesial, midfacial, distal, palatal).

Soft tissue dimensions were measured as follows:

1. Papilla levels. The levels were recorded by means of an acrylic stent provided with direction grooves.

Papilla level (mesial and distal) was defined as the distance from the top of the groove to the top of the papilla measured to the nearest 0.5 mm using a manual probe (CP 15 UNC, Hu-Friedy ® , Chicago, USA).

2. Midfacial mucosa level. The level of the peri-implant mucosa at the midfacial aspect of the tooth/restoration was measured using the same acrylic stent provided with a central direction groove.

The midfacial level was defined as the distance from the top of the groove to the first contact with the peri-implant mucosa measured to the nearest 0.5 mm using a manual probe (CP 15 UNC, Hu-Friedy ® , Chicago, USA). Given the aforementioned research hypothesis, midfacial mucosa level was considered the primary outcome variable of the study.

All soft tissue dimensions were recorded by two clinicians (JC/TDR). The results on the inter-examiner reliability can be found in an earlier paper (De Rouck et al. 2008a).

Aesthetic outcome

All aesthetic evaluations relating to the soft tissues and implant crowns were performed by one clinician (AE) who had not been involved in any treatment. This clinician was calibrated prior to the study on the basis of 20 single implant cases in the anterior maxilla. Per case a frontal and occlusal colour slide was available and each case was scored twice with an interval of one week. The 20 single implant cases were also scored by another clinician (JC). The results on the intra-and inter-examiner reliability can be found in a recent paper [START_REF] Cosyn | Single implant treatment in healing versus healed sites of the anterior maxilla: an aesthetic evaluation[END_REF].

The Pink Esthetic Score (PES) by [START_REF] Fürhauser | Evaluation of soft tissue around single-tooth implant crowns: the pink esthetic score[END_REF] was used to evaluate the aesthetic outcome of the periimplant soft tissues. This index includes 7 variables: mesial papilla, distal papilla, midfacial level, midfacial contour, alveolar process deficiency, soft tissue colour and soft tissue texture. Each parameter is assessed with a The White Esthetic Score (WES) by [START_REF] Belser | Outcome evaluation of early placed maxillary anterior single-tooth implants using objective esthetic criteria: a cross-sectional, retrospective study in 45 patients with a 2-to 4-year follow-up using pink and white esthetic scores[END_REF] was used to evaluate the aesthetic outcome of the visible part of the implant restoration. This index includes 5 variables: tooth form, tooth volume, tooth colour including the assessment of hue and value, tooth texture and translucency. Again, each parameter is assessed with a 0-1-2 score with 2 being the best and 0 being the worst score. Thus, a maximum score of 10 can be reached. All variables are assessed by comparison with a reference tooth which is the contralateral tooth for incisor and cuspid replacements and the neighbouring premolar for premolar replacements. The authors set the threshold for clinical acceptance arbitrarily at 6/10. A score of 9/10 or more was considered (almost) perfect.

The overall aesthetic outcome was assessed by combining the results of the PES and WES. If PES ≥ 12 and WES ≥ 9, the treatment was considered (almost) perfect. If PES < 8 and/or WES < 6, the result was considered a failure.

Statistical analysis

Data analysis was performed using the patient as the experimental unit. For all parameters mean values were calculated, if applicable. Descriptive statistics also included frequency distributions for papillae and midfacial mucosa level. The changes between the 1-and 3-year reassessment were examined using the Wilcoxon signed ranks test. The level of significance was set at 0.05.

Results

From the 32 patients who had been scheduled from May 2005 to June 2006, 30 (14 men, 16 women; mean age of 54 with a range from 24 -76) were treated for single-tooth replacement in the aesthetic zone by means of an immediate implant (Nobelreplace tapered TiUnite ® , Nobel Biocare, Göteborg, Sweden). Two patients had to be excluded during surgery as loss of the buccal bone crest had occurred after tooth extraction. For details on the reasons for tooth loss, implant locations and dimensions, we wish to refer to an earlier paper (De Rouck et al. 2008a). During the 3-year observation period, 1 and 3 patients were lost to follow-up after 3 and 12 months, respectively. One of them moved and 1 could not be contacted even after several attempts. The other 2 patients agreed to come in for evaluation but did not show up in the end.

Implant survival and complications

At one month follow-up, one of the implants was found to be mobile causing pain and discomfort (tooth location 21; diameter 5 mm -length 16 mm). Besides this one early failure, all implants remained well-integrated resulting in a 96 % implant survival rate after 3 years of function. With respect to complications, one permanent crown lost retention at 8 months of follow-up and was re-cemented. There were no other technical, nor biologic complications.

Hard tissue parameters

Table 1 shows mean bone loss from baseline (implant installation) at the 1-and 3-year reassessment. Bone loss significantly increased between these intervals (p ≤ 0.038). After 3 years of function radiographic examination yielded on average 1.13 mm mesial, respectively 0.86 mm distal bone loss. Hence, the mean overall bone loss was 1.00 mm.

(HERE APPROXIMATELY TABLE 1 PLEASE)

Soft tissue parameters

In table 2 the clinical conditions of the implant restorations are shown. Throughout the study period, mean plaque levels remained low (< 20 %) indicating good oral hygiene. Between 1 and 3 years, a significant reduction in probing depth from 3.46 mm to 3.17 mm (p = 0.015) occurred coinciding with a significant bleeding on probing drop from 41 % to 24 % (p = 0.002).

(HERE APPROXIMATELY TABLE 2 PLEASE)

Table 3 depicts the dimensional changes of the soft tissue outline around the implant restorations in relation to the status prior to tooth extraction. Mesial papillae showed a significant re-growth between 1 and 3 years (p = 0.015) pointing to a mean loss of only 0.05 mm from the pre-operative status at study termination. A similar trend was found for distal papillae (p = 0.117) resulting in a final mean loss of 0.08 mm. At 3 years follow-up severe mesial papilla loss (> 1 mm) was found in 1/25 (4 %) and severe distal papilla loss (> 1 mm) in 4/25 (16 %) cases. In 13/25 (52 %) patients mesial papillae regained at least their original height. In 14/25 (56 %) patients distal papillae regained at least their original height. The midfacial mucosa level did not alter significantly between the 1-and 3-year reassessment (p = 0.135). At study termination a mean recession from the pre-operative status of 0.34 mm was found (table 3). At 3 years follow-up advanced midfacial recession (> 1 mm) was found in 2/25 (8 %) cases. In 10/25 (40 %) patients the midfacial mucosa regained at least its original level.

A case is shown in figure 1 illustrating papillary re-growth and stability of the midfacial mucosa level between the 1-and 3-year reassessment.

(HERE APPROXIMATELY TABLE 3, FIGURE 1 PLEASE)

Aesthetic outcome

Table 4 shows the results of all 7 criteria of the PES. Midfacial level and soft tissue colour were most satisfying showing a perfect match with the corresponding tooth in 17/25 (70 %) cases. Unfavourable results were most prevalent for the alveolar process showing severe deficiency in 5/25 (20 %) cases.

The mean PES was 10.48 (SD 2.47; range 5 -14). Figure 2 shows the cumulative percent of the PES. Dotted lines indicate the upper limit for an unacceptable result (PES = 7) and a favourable, yet imperfect result (PES = 11). Four out of 25 (16 %) cases showed an unfavourable outcome and 9/25 (36 %) an (almost) perfect result.

(HERE APPROXIMATELY TABLE 4 & FIGURE 2 PLEASE)

Table 4 shows the results of all 5 criteria of the WES. Since 1 patient refused to replace the provisional restoration, the results on the WES were based on 24 cases.

Tooth texture was most satisfying indicating an ideal result in 21/24 (88 %) cases. Unfavourable results were most prevalent for tooth colour with a severe mismatch in 5/24 (21 %) and a perfect result in only 9/24 (38 %) cases.

The mean WES was 8.17 (SD 1.52; range 5 -10). Figure 3 shows the cumulative percent of the WES. Dotted lines indicate the upper limit for an unacceptable result (PES = 5) and a favourable, yet imperfect result (PES = 8). Two out of 24 (8 %) cases showed an unfavourable outcome and 12/24 (50 %) an (almost) perfect result.

(HERE APPROXIMATELY FIGURE 3 single implant treatments showed an (almost) perfect result (PES ≥ 12 and WES ≥ 9). An acceptable result (PES: 8 -11 and WES: 6 -8) was found for 14/24 (58 %) cases. The aesthetic outcome was unfavourable for 5/24 (21 %) single implant treatments. Three of them (13 %) were considered unfavourable because of a PES < 8, another one (4 %) because of a WES < 6. One case (4 %) showed a PES < 8 and WES < 6 and could be regarded as a complete aesthetic failure.

Discussion

In the present study 96 % of the implants survived and mean bone loss was 1 mm after 3 years of function. These data correspond well with the existing knowledge on survival and bone remodelling of conventionally installed single TiUnite ® (Nobel Biocare, Göteborg, Sweden) implants after a comparable observation period (Turkyilmaz et al. 2007, De Bruyn et al. 2009, Eghbali et al. 2010). Hence, the timing of implant placement relative to tooth extraction does not seem to be decisive for implant survival or bone remodelling, which has also been demonstrated by at least 4 controlled clinical studies using various implant systems [START_REF] Lindeboom | Immediate placement of implants in periapical infected sites: a prospective randomized study in 50 patients[END_REF], Palatella et al. 2008[START_REF] Block | Prospective evaluation of immediate and delayed provisional single tooth restorations[END_REF], Van Kesteren et al. 2010).

As described in a systematic review on single implants in the anterior maxilla [START_REF] Den Hartog | Treatment outcome of immediate, early and conventional single-tooth implants in the aesthetic zone: a systematic review to survival, bone level, soft-tissue, aesthetics and patient satisfaction[END_REF], few reports included data on clinical parameters. In our study plaque levels were fairly low and comparable between the 1-and 3-year reassessment. In contrast, probing depth and bleeding on probing significantly reduced within this time frame. This trend had already started in the first year (De Rouck et al. 2008a). Similar observations have been described in other short- [START_REF] Proussaefs | Effects of immediate loading with threaded hydroxyapatite-coated root-form implants on single premolar replacements: a preliminary report[END_REF] and long-term studies [START_REF] Apse | The longitudinal effectiveness of osseointegrated dental implants. The Toronto Study: peri-implant mucosal response[END_REF].

Even though a number of reports are available on immediate single implant treatment, few prospective studies have been published with data on soft tissue dynamics (Kan et al. 2003a, De Rouck et al. 2008a, Palatella et al. 2008[START_REF] Block | Prospective evaluation of immediate and delayed provisional single tooth restorations[END_REF], Van Kesteren et al. 2010[START_REF] Raes | Immediate and conventional single implant treatment in the anterior maxilla: one-year results of a case series on hard and soft tissue remodelling and aesthetics[END_REF]). However, these papers all described shortterm results with observations up to 2 years. The results of the present 3-year study showed that papillae had not fully remodelled after 1 year of function. Significant papillary re-growth was observed especially at the mesial aspect and at the 3-year reassessment papillae had basically regained their original height. These findings indicate that the presence of papillae may not be the key issue following immediate single implant treatment providing these were intact at the time of tooth loss. Similar observations have been described following conventional implant surgery [START_REF] Jemt | Regeneration of gingival papillae after single-implant treatment[END_REF][START_REF] Choquet | Clinical and radiographic evaluation of the papilla level adjacent to single-tooth dental implants. A retrospective study in the maxillary anterior region[END_REF], Kan et al. 2003b[START_REF] Henriksson | Measurements of soft tissue volume in association with single-implant restorations: a 1-year comparative study after abutment connection surgery[END_REF], An issue that gained at lot of attention in recent studies relates to the midfacial mucosa level following immediate single implant treatment [START_REF] Lindeboom | Immediate placement of implants in periapical infected sites: a prospective randomized study in 50 patients[END_REF][START_REF] Chen | A prospective clinical study of non-submerged immediate implants: clinical outcomes and esthetic results[END_REF][START_REF] Juodzbalys | Soft and hard tissue assessment of immediate implant placement: a case series[END_REF][START_REF] Kan | Effects of the facial osseous defect morphology on gingival dynamics after immediate tooth replacement and guided bone regeneration: 1-year results[END_REF], De Rouck et al. 2008a[START_REF] Evans | Esthetic outcomes of immediate implant placements[END_REF], Palatella et al. 2008[START_REF] Block | Prospective evaluation of immediate and delayed provisional single tooth restorations[END_REF], Chen et al. 2009, Redemagni et al. 2009, Van Kesteren et al. 2010[START_REF] Raes | Immediate and conventional single implant treatment in the anterior maxilla: one-year results of a case series on hard and soft tissue remodelling and aesthetics[END_REF]. According to a review article immediate implants show an increased risk for advanced midfacial recession (> 1 mm) [START_REF] Chen | Clinical and esthetic outcomes of implants placed in postextraction sites[END_REF]). However, only 2/25 (8 %) of our cases demonstrated more than 1 mm recession and mean loss was only 0.34 mm after 3 years without deterioration between the 1-and 3-year reassessment. In addition, of all the criteria of the PES midfacial level was most satisfying. On the basis of these observations, the risk for advanced midfacial recession was clearly low in our patients, hereby confirming the aforementioned research hypothesis. Interestingly, our findings contrast the conclusion of a review article by [START_REF] Chen | Clinical and esthetic outcomes of implants placed in postextraction sites[END_REF], yet seem to be in agreement with 3 controlled clinical studies describing a low risk for advanced midfacial recession following immediate single implant treatment (Palatella et al. 2008[START_REF] Block | Prospective evaluation of immediate and delayed provisional single tooth restorations[END_REF], Van Kesteren et al. 2010). This contradiction is interesting and may be explained by disparities in study design (prospective versus retrospective), recording procedure (using a stent or standardized digital slides with fixed reference points or not), case selection (thinscalloped biotype cases excluded or not; buccal bone crest intact or not), surgical aspects (surgeon's experience; implant type; orofacial implant positioning; hard and/or soft tissue grafting or not) and restorative aspects (immediate provisionalisation or not). Given the complexity of this aspect of treatment outcome, a thorough systematic review would be valuable specifically comparing the risk for advanced midfacial recession between immediate and conventional single implant treatment.

Aesthetic aspects relating to the soft tissues and implant crown are more and more becoming the key for success.

Although ample reports have been published on immediate implantation and restoration, few have documented the aesthetic outcome using objective criteria [START_REF] Juodzbalys | Soft and hard tissue assessment of immediate implant placement: a case series[END_REF], Chen et al. 2009[START_REF] Raes | Immediate and conventional single implant treatment in the anterior maxilla: one-year results of a case series on hard and soft tissue remodelling and aesthetics[END_REF].

Since these papers all described short-term results with observations up to 1 year we believe this 3-year paper may add relevant information. When scrutinizing the results on the PES, 16 % of all cases showed an unfavourable outcome (PES ≤ 7), which is slightly higher when compared to earlier findings in the short term (0 % -11 %) [START_REF] Juodzbalys | Soft and hard tissue assessment of immediate implant placement: a case series[END_REF], Chen et al. 2009[START_REF] Raes | Immediate and conventional single implant treatment in the anterior maxilla: one-year results of a case series on hard and soft tissue remodelling and aesthetics[END_REF]. On the other hand, 36 % of our cases showed an (almost) perfect outcome (PES ≥ 12), which resembles quite well with available data (26 % -39 %) [START_REF] Juodzbalys | Soft and hard tissue assessment of immediate implant placement: a case series[END_REF], Chen et al. 2009[START_REF] Raes | Immediate and conventional single implant treatment in the anterior maxilla: one-year results of a case series on hard and soft tissue remodelling and aesthetics[END_REF]. To our knowledge only 4 case series have been published documenting the aesthetic characteristics of single implants crowns [START_REF] Belser | Outcome evaluation of early placed maxillary anterior single-tooth implants using objective esthetic criteria: a cross-sectional, retrospective study in 45 patients with a 2-to 4-year follow-up using pink and white esthetic scores[END_REF], Buser et al. 2009, Cosyn et al. 2010[START_REF] Raes | Immediate and conventional single implant treatment in the anterior maxilla: one-year results of a case series on hard and soft tissue remodelling and aesthetics[END_REF]). In the present study 8 % of the cases could be considered failures in this respect (WES ≤ 5), which falls within the range described in the literature (0 % -21 %) [START_REF] Belser | Outcome evaluation of early placed maxillary anterior single-tooth implants using objective esthetic criteria: a cross-sectional, retrospective study in 45 patients with a 2-to 4-year follow-up using pink and white esthetic scores[END_REF], Buser et al. 2009, Cosyn et al. 2010[START_REF] Raes | Immediate and conventional single implant treatment in the anterior maxilla: one-year results of a case series on hard and soft tissue remodelling and aesthetics[END_REF]). In addition, 50 % of our cases showed an (almost) perfect result which is in agreement with others [START_REF] Buser | Early implant placement with simultaneous guided bone regeneration following single-tooth extraction in the esthetic zone: 12-month results of a prospective study with 20 consecutive patients[END_REF], Cosyn et al. 2010[START_REF] Raes | Immediate and conventional single implant treatment in the anterior maxilla: one-year results of a case series on hard and soft tissue remodelling and aesthetics[END_REF]), yet in contrast with [START_REF] Belser | Outcome evaluation of early placed maxillary anterior single-tooth implants using objective esthetic criteria: a cross-sectional, retrospective study in 45 patients with a 2-to 4-year follow-up using pink and white esthetic scores[END_REF] showing perfection in only 18 % of the cases. In this regard it must be emphasized that patients had been sent back to the referring dentist for restorative treatment.

Of particular importance is the overall aesthetic outcome combining the results of the PES and WES. As such, 21 % showed perfection (PES ≥ 12 and WES ≥ 9) which is quite modest, yet in agreement with the current knowledge on single implant treatment (7 % -35 %) [START_REF] Belser | Outcome evaluation of early placed maxillary anterior single-tooth implants using objective esthetic criteria: a cross-sectional, retrospective study in 45 patients with a 2-to 4-year follow-up using pink and white esthetic scores[END_REF], Buser et al. 2009, Cosyn et al. 2010[START_REF] Raes | Immediate and conventional single implant treatment in the anterior maxilla: one-year results of a case series on hard and soft tissue remodelling and aesthetics[END_REF]. Similarly, 21 % of our cases could be considered aesthetic failures (PES < 8 and/or WES < 6) which also falls within the range of what has been published (5 % -34 %) [START_REF] Meijndert | Evaluation of aesthetics of implant-supported single-tooth replacements using different bone augmentation procedures: a prospective randomized clinical study[END_REF][START_REF] Belser | Outcome evaluation of early placed maxillary anterior single-tooth implants using objective esthetic criteria: a cross-sectional, retrospective study in 45 patients with a 2-to 4-year follow-up using pink and white esthetic scores[END_REF], Buser et al. 2009, Cosyn et al. 2010[START_REF] Raes | Immediate and conventional single implant treatment in the anterior maxilla: one-year results of a case series on hard and soft tissue remodelling and aesthetics[END_REF]. Clearly, optimal aesthetics seem difficult to achieve and failures are quite prevalent in spite of the fact that patients had been selected on the basis of stringent criteria and treated by experienced clinicians. It seems wise to warn patients for this relatively high risk.

In conclusion, the results of this 3-year prospective study indicate that immediate single implant treatment may be considered a valuable and predictable option in terms of implant survival and hard and soft tissue remodelling. We observed in a 3-year time span nearly full papillary re-growth and a low risk for advanced midfacial recession with 19/24 (79 %) cases showing acceptable to excellent aesthetics. In this context, we wish to emphasize the favourable starting point in all cases excluding high-risk patients with a thin-scalloped gingival biotype and/or buccal bone defect.

  soft tissue level/contour at the facial aspect of the failing tooth in perfect harmony with the surrounding teeth.5. Thick gingival biotype as determined by De Rouck et al. (2009b).6. Adequate bone height apical to the alveolus of the failing tooth (≥ 5 mm) to ensure primary implant stability of at least 35 Ncm.Exclusion criteria were as follows:1. Systemic diseases.2. Smoking.3. Bruxism, lack of posterior occlusion.4. Non-treated periodontal diseases.5. Presence of active infection (pus, fistula) around the failing tooth.6. Loss of the buccal bone crest after extraction of the failing tooth.

  for completeness; the other variables are assessed by comparison with a reference tooth which is the contralateral tooth for incisor and cuspid replacements and the neighbouring premolar for premolar replacements. The authors set the threshold for clinical acceptance arbitrarily at 8/14. A score of 12/14 or more was considered (almost) perfect.

  outcome was assessed by combining the results of the PES and WES. Five out of 24 (21 %)

  between a single implant and tooth.

  Changes in soft tissue dimensions in relation to the pre-operative status at 1 ) [range]; negative value indicates recession in relation to the pre-operative status

Fig. 1 .

 1 Fig.1. One-year and 3-year result of a single-implant case.

Table 1 .

 1 Besides careful case selection, appropriate surgical and restorative procedures and clinical experience are also considered of pivotal importance. More prospective studies monitoring soft tissue dynamics over longer time periods are needed. Since it is currently unclear to what extent treatment modalities influence aesthetics, comparative studies specifically focusing on this aspect of treatment outcome would be valuable. Marginal bone loss in relation to baseline (implant installation) at 1 and 3 years

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 .

 2 Clinical conditions at 1 and 3 years

	Location	1 year (n = 28)	3 years (n = 25)	p-value
	Plaque score (%)	17 (18) [0; 50]	18 (17) [0; 50]	0.668
	Probing depth (mm)	3.46 (0.69) [2.00; 4.70]	3.17 (0.63) [2.00; 4.30]	0.015
	Bleeding on probing (%)	41 (16) [0; 75]	24 (19) [0; 50]	0.002
	n number of patients
	Mean (SD) [range]

Table 4 .

 4 Aesthetic outcome at 3 years

	Parameter	0	1	2
	Mesial papilla	2	10	13
	Distal papilla	4	8	13
	Midfacial level	2	6	17
	Midfacial contour	2	11	12
	Alveolar process deficiency	5	4	16
	Soft tissue colour	1	7	17
	Soft tissue texture	1	8	16
	Pink Esthetic Score (n = 25)	10.48 (2.47) [5-14]
	Mean (SD) [range]
	Tooth form	0	10	14
	Tooth volume	0	5	19
	Tooth colour	5	10	9
	Tooth texture	0	3	21
	Translucency	1	4	19
	White Esthetic Score (n = 24)	8.17
	Mean (SD) [range]

Journal of Clinical Periodontology -PROOFJournal of Clinical Periodontology -PROOF