The Strategic Scorecards -An instrumentation of the strategic management accounting

Exploration of a concept, instrumentation and results from a french empirical study

I describe in this study a generic model of strategic management accounting instrumentation: the strategic scorecards. In order to build this generic model, I review in the existing literature the concepts of Strategic Control and Strategic Management Accounting (SMA). I present the characteristics of these concepts and analyse the reasons why they emerged. I show how the strategic scorecards are an instrumentation of the SMA in studying the most widely known and esteemed scorecards: the Balanced Scorecards (Kaplan & Norton, 1996) and Skandia's Navigator [START_REF] Edvinsson | Intellectual Capital, Realizing your company's true value by finding its hidden brainpower[END_REF]. I then clarify the outlines for a generic model of strategic scorecard and disclose an "a priori" typology of this instrument. I investigate the interest for Scorecards from french managers through an empirical research based on a questionnaire. Using a typological analysis, I present five categories of strategic scorecards and the characteristics of the companies associated with the different types of scorecards. Finally, I present results of fifteen interviews confirming the previous classification. French managers underlined some problems in integrating the operational and strategic dimensions of management accounting. In conclusion, french management accounting evolves to a more strategic and external conception.

CONCEPT OF STRATEGIC MANAGEMENT ACCOUNTING AND UNDERLYING INSTRUMENTS

Although sharing most of the same fundamental assumptions, the two concepts of Strategic Control and Strategic Management Accounting have developed two separate fields of management tools. Both underline the strong interrelations existing between Strategic Management and Management Control. In order to be an efficient decision tool, a strategic control system must closely follow each step of the implementation of the strategy and the achievement of pre-defined objectives.

FROM MANAGEMENT CONTROL TO STRATEGIC CONTROL AND STRATEGIC MANAGEMENT ACCOUNTING

The conventional developments in the field of management control [START_REF] Johnson | Relevance Lost: The rise and fall of management accounting[END_REF] disclose an opposition among the process of strategic management, the process of management control and the process of operational control. Moreover strategic control and strategic management accounting are focused on the existing relations between strategic management and management control. For Anthony (1965:17), management control is "the overall of the financial and accounting tools of check on the basis of predetermined objects", in other words, a process of planning verification. The concept of strategic control emerged during the 1970's and has been developed since [START_REF] Sarrazin | Strategic Control: A Normative Theory of Corporate Development Under Ambiguous Circumstances[END_REF][START_REF] Schendel | Strategic Management: A New View of Business Policy and Planning[END_REF][START_REF] Horovitz | Strategic Control: a new task for top management[END_REF]. Some authors described the relations between strategic planning and budget making [START_REF] Vancil | What kind of management control do you need ?[END_REF][START_REF] Lorange | A framework for Management Control Systems[END_REF][START_REF] Lorange | How to design a strategic planning system[END_REF]. [START_REF] Newman | Constructive Control -Design and Use of Control Systems[END_REF] presented a more constructive and future or "forward" oriented model. The concept of strategic management accounting was formulated in the beginning of the 1980's [START_REF] Simmonds | Strategic Management Accounting[END_REF]. Researches on this subject have kept increasing since then (Shank& Govindarajan, 1990;[START_REF] Bromwich | The case for strategic management accounting: the role of accounting information for strategy in competitive markets[END_REF].

The most significant differences between the two concepts are the applied and operational purposes. This may be explained by the difference of scientific culture of the authors from both fields. Academics in strategic control are all strategic management professors and academics in strategic management accounting are mainly management control researchers. I believe that this divergence is a useful source for management tools critical thinking.

After reviewing the different definition of strategic control and strategic management accounting, I propose a typology of the different versions of strategic management accounting (terminology now used for both works in the field of strategic control and works in the field of strategic management accounting). Three versions are distinguished.

-a simple or minimalist version of strategic management accounting: management control is a tool to strategy formulation [START_REF] Camillus | Operational planning: the integration of programming and budgeting[END_REF][START_REF] Simmonds | Strategic Management Accounting[END_REF][START_REF] Lorange | Strategic Control[END_REF][START_REF] Bromwich | The case for strategic management accounting: the role of accounting information for strategy in competitive markets[END_REF]. Management control proceeds, in this case, of an external vision of the company (Teller, 1999:91). -A median version : management control helps to build the corporate strategy [START_REF] Shank | Strategic cost analysis: the evolution from managerial to strategic accounting[END_REF]& 1990;[START_REF] Wilson | Strategic Management Accounting[END_REF][START_REF] Band | Strategic Control through Core Competencies[END_REF][START_REF] Oldman | Cost management and its interplay with business strategy and context[END_REF]. This version captures the management control based on the competitive advantage theory. -A large version. Management accounting is itself a component in the formulation of the strategy (Simons 1990;[START_REF] Band | Strategic Control through Core Competencies[END_REF][START_REF] Teller | Le contrôle de gestion -Pour un pilotage intégrant stratégie et finance [Management accounting: for a driving which integrates strategy and finance[END_REF]. This version is, so far, the most innovative approach in strategic management accounting.

The principal reasons to implement a strategic management accounting process given in the literature lie in the evolution of the environment. This evolution is described in successive stages: stable & predictable to, unstable & difficult to anticipate, to turbulent & unpredictable. As a consequence, control tools must include external and leading indicators (strategic, prospective) and integrate them into the navigation of the company. Those indicators have to be articulated with the traditional internal financial indicators. Some other considerations are presented in the literature but are not developed here. In my opinion, strategic management control receives much attention due to human resources management and to the increasing complexity of organizations decisional processes.

THEORETIC EXPLORATION OF THE STRATEGIC DIMENSION OF STRATEGIC MANAGEMENT ACCOUNTING PROCESSES

Types of management control associated to adaptive strategies

Nature of the relation between strategic management and management accounting.

Two trends characterize the literature in Strategy [START_REF] Lauriol | La stratégie et ses représentations dans les ouvrages de langue française (1990-1996)[END_REF]. The first one focuses on the company in its environment. The second one focuses on the study of the processes of strategy formulation. In the first trend, I distinguish the adaptative strategies to the proactive strategies. The adaptative strategies belong to the competitive advantage approach [START_REF] Porter | Competitive Advantage: Creating and Sustaining Superior Performance[END_REF] which has its origins in the industrial economic theory. In contrast, the proactive strategies break with the standard industrial economic theories [START_REF] Boissin | La structuration de la recherche francophone en stratégie: une analyse bibliographique[END_REF].

As [START_REF] Teller | Le contrôle de gestion -Pour un pilotage intégrant stratégie et finance [Management accounting: for a driving which integrates strategy and finance[END_REF] explains, the adaptative strategies (SWOT model: strengths-weaknesses, opportunities-threats) are designed to a type of management control focused on the financial and shareholder value or on the competitive advantage. Standard management control is based on an "heteronomous" or deliberated approach to the corporate strategy (adaptative strategy) in the trend that studies the company in its environment. In this classical approach management control stays dependent on the strategy in a one-way relation.

Most of the authors in strategic management accounting rely on this approach of the strategy (Teller 1999:59) as well as all the works classified in the first and second version of the strategic management accounting (see Table 1). In these models, the relation between the strategy and the control is thought excluding operationalization (action). Nowadays, it becomes more difficult to decline the strategy at an action level (see the developments about complexity) although, this was exactly the first role assigned to management control [START_REF] Johnson | Relevance Lost: The rise and fall of management accounting[END_REF].

Strategic management models which make possible an interactive relation between Strategy and Control.

The Resource Based View (Laroche & Nioche, 1998: 135-65) lies within the scope of the evolutionist theories. The evolutionist theory postulates that managing the technical and organizational processes evolution builds the competitive position. The Resources Based View approach is based on 5 main hypotheses. First, the organizational processes generate a set of routines. Second R&D play a major role according to their capacity to modify the routines. Third, actors are subjects to bounded and procedural rationality, hence the interest of the advocates for the RBV theory for the processes of organizational learning. Fourth, every organization has an idiosyncratic character. Fifth, the company is supposed to evolve in an uncertain environment within which the markets of production factors are incomplete and imperfect.

The different evolutionist authors distinguish the notion of resources from the one of competencies. Resources are defined as discrete strategic assets (individual know-how, production capacities) and competencies as strategic assets allowing the implementation of other production factors (collective know-how and capacity to coordinate several production processes). Instead of distinguishing the resources from the competencies, some typologies allocate them between tangible & intangible assets. However, few of them mention the existing interrelations between the resources and the competitive advantage. [START_REF] Hall | A framework linking intangible resources and capabilities to sustainable competitive advantage[END_REF] suggests a different classification. He separates the resources which depend on individuals (for example reputation) from those which don't (for example databases). He makes the distinction between the capacities (or abilities) of the company, based on assets, from those based on competencies. Finally, he associates a capacity (weighting) to every intangible resource. This allows to characterize more precisely the strategic resources. Within the framework of the RBV, the classic strategic process is reversed (Grant, 1991: 116). It consists at first to proceed to an internal analysis that allows to identify the strategic assets, then, to measure and characterize the resources and competencies. In the end, the method suggests to operate an external analysis in including the identified resources and competencies.

The major critic addressed to the RBV [START_REF] Shay | Dynamic Competitive Strategy: Towards a Multiperspective Conceptual Framework[END_REF] relates to the weakness of the dynamic of the RBV frame because of a lack of analysis of the competitive system. The models issued from the RBV do not analyse the competitive system and therefore renders difficult to separate the most important resources at a specific time. In my opinion, the argument stating the weakness of the dynamic is due to the dissociation of the two sequences of the strategy analysis: first the process of strategy formulation and second the competitive positioning. Yet, such approach becomes less relevant as the environment gets more turbulent and complex where any projected analysis and any planning can be made.

A third trend exists between the "heteronomous" strategies (deliberated strategies) and the voluntarist strategies (emergent strategies): it consists in adapting the strategy as soon as it is implemented. [START_REF] Mintzberg | Of Strategies, Deliberate and Emergent[END_REF] name this trend " the process strategy". They explain (1985:270) how the formulation originate within the processes. They are at the same time deliberate and emergent.

In France, [START_REF] Avenier | La Stratégie Chemin-Faisant [The Strategy on the Way[END_REF] calls this approach "the strategy along the way" and Koening (1996) "the interactive strategy". It seems to me relevant to apply the models stemming from the RBV with the framework of an interactive strategy conception. This conception consists in defining a "mission" clarified with objectives. These objectives would appear gradually from the confrontation of the vision of managers to the real events. The large version of the strategic management accounting requires an interactive strategic process. Teller (1999:55) explains that an innovative version of strategic management accounting needs an interrelation between the formulation and the implementation of the strategy. Therefore, the interactive strategy process is inspired, in different levels, from heteronomous and voluntarist strategies, according to the characteristics of the company and its environment.

SMA developments likely to be associated with the heterodox approaches in strategic management.

Only a few authors have proposed significant strategic management accounting developments using heterodox approaches to Strategy. [START_REF] Newman | Constructive Control -Design and Use of Control Systems[END_REF] suggested the adoption of a control process, called "the steering control". He explained how managers must define goals accepted by workers. He emphasizes on the participation of employees to secure understanding and acceptance to this management control process. [START_REF] Lorange | Strategic Control: some issues in making it operationnally useful[END_REF][START_REF] Lorange | Strategic Control[END_REF] proposed the notion of "strategic leap control" designed to turbulent environments. Using the strategic leap control, the objectives are modified along with the strategy. More explicitly, Simons (1990Simons ( , 1992) ) developed the concept of "interactive control systems" which collects data on strategic uncertainties. This type of control needs "not to focus on the implementation of intended strategies, but rather on the formation of tomorrow's strategy" (1992:48).

THREE VIEWS ON THE STRATEGIC MANAGEMENT ACCOUNTING

Strategic management accounting is based on a process approach of management accounting.

The company is a network of horizontal, flat and transverse structures where the activities are organized according to market imperatives. The development at the bottom of the process constitutes a fundamental driver to integration. In particular the ABC method (Activity Based Costing), ABM (Activity based Management) and ABB (Activity Based Budgeting) represent competencies-based tools. I conclude on the existence of a significant relation between the processes and the competencies of a company, therefore, that a widened approach of strategic management accounting needs a RBV approach of the strategy. The third version of SMA is based on a "core competencies" approach [START_REF] Hamel | Competing for the future[END_REF]. It supposes both strategic and financial analysis, a strategic process extended to stakeholders (customers, suppliers, employees, community, etc…). To sum up, four types of management control can be distinguished:

-the management control with the financial focus. The principal tools are the conventional budgets and reporting sheets. These tools use exclusively financial data and are limited to a one year period. If the period exceeds one year, the tools of strategic management are more adapted.

-Two versions of the strategic management accounting: a minimalist version and a median version. Researchers in the field of strategic control have designed new tools including the strategic budgets and operational budgets dimensions [START_REF] Camillus | Operational planning: the integration of programming and budgeting[END_REF][START_REF] Lorange | Strategic Control: some issues in making it operationnally useful[END_REF][START_REF] Lorange | Strategic Control[END_REF]. But the restrictive nature of these instruments remains. They belong the minimalist version. For the median version, researchers in strategic management accounting consider that budgets have to be completed with other approaches. [START_REF] Simmonds | Strategic Management Accounting[END_REF] & [START_REF] Bromwich | The case for strategic management accounting: the role of accounting information for strategy in competitive markets[END_REF] suggest to use qualitative and external measures with three dimensions of analysis: the products & customers dimension, the competitive dimension and the environmental dimension. [START_REF] Shank | Strategic cost analysis: the evolution from managerial to strategic accounting[END_REF] have developed an operational model with the definition of Key Success Factors, determined in using a competitive analysis of the environment and an analysis of the internal processes of the company, with the help of the ABC method. -The large version called the Integrated SMA. Strategic control and strategic management accounting specialists have proposed no significant developments.

The table 1 describes the typology of the SMA.

THE BALANCED SCORECARD, INSTRUMENTATION OF THE MEDIAN VERSION OF THE SMA

The BSC (Kaplan & Norton, 1996) is an example of strategic scorecard. Figure 1 summarizes the main characteristics of this tool. On many aspects, the BSC responds to the characteristics of a strategic management accounting tool. Teller (1999: 220) believes that the BSC is an instrumentation of SMA for the following reasons:

-the BSC is a process approach to the organization, -the strategic and financial analyses are equally considered, -and the BSC aims to be a global corporate performance analysis (financial analysis, customer analysis, processes & innovation analysis, organizational learning analysis). However, a close review of [START_REF] Kaplan | The Balanced Scorecard -Measures That Drive Performance[END_REF], 1993, 1996a, 1996b) leads to the conclusion that the BSC does not fulfill all the principles of the large version of SMA:

-use of traditional approach to strategy (SWOT model, 1998: 62-7), -the formulation and implementation of the strategy are separate steps, -the strategic value is fundamentally based on customer satisfaction and depends on the financial value.

Table 1. Synthesis about the interrelations of the strategic processes and the management control processes.

The minimalist version of the SMA is embodied in the Competitive Scorecards (PIMS program: [START_REF] Anderson | PIMS: A Reexamination[END_REF]Noon & Bates, 1993) and in the stakeholders Scorecards (Atkinson, 1997).

The BSC is more comprehensive than those 2 models because it insists on a balanced perspective of external and internal analysis. The BSC has also been conceived on the basis of the following 3 principles of SMA (Skank & Govindarajan, 1989): -a strategic approach based on the principles of competitive advantage [START_REF] Porter | Competitive Advantage: Creating and Sustaining Superior Performance[END_REF], -a processes approach to the organization, implying the use of ABC and ABM methods by management control, -and Key Success Factors establishment, very close to Kaplan & Norton's leading indicators. I conclude that the BSC fulfills the principles of the median version of the SMA. Kaplan and Norton, 1996).

THE INTELLECTUAL CAPITAL SCORECARD: AN INSTRUMENTATION OF THE LARGE VERSION OF THE SMA ?

The models of intellectual capital scorecards [START_REF] Roos | Measuring your Company's Intellectual Performance[END_REF] propose another type of Strategic Scorecards. The Navigator, conceived by the swedish insurance company Skandia, (see figure 2), is the most widely known and complete intellectual capital scorecard [START_REF] Edvinsson | Intellectual Capital, Realizing your company's true value by finding its hidden brainpower[END_REF]. Still, these instruments remain derived from the BSC. Their specificity, according to their designers [START_REF] Sveiby | The New Organizational Wealth -Managing and Measuring Knowledge-Based Assets[END_REF][START_REF] Edvinsson | Intellectual Capital, Realizing your company's true value by finding its hidden brainpower[END_REF]) is to allow an analysis of the intangible resources. They are designed according to the RBV theory [START_REF] Roos | Measuring your Company's Intellectual Performance[END_REF]. The Navigator separates the Human Capital (Knowledge, know-how, attitude, behavior and intellectual agility) to the Structural capital (organization, relations with partners, renewal and development). It seems to me possible to draw a parallel between this typology of the intellectual capital and the one presented by [START_REF] Hall | A framework linking intangible resources and capabilities to sustainable competitive advantage[END_REF]. Hall separates the organization capacities based on employees competencies (human capital) to other assets non-based on employees (structural capital). Despite several differences, the Navigator and the Intellectual capital scorecards have both been conceived in the frame of the RBV. Moreover, [START_REF] Grant | The Resource-Based Theory of Competitive Advantage: Implications for Strategy Formulation[END_REF], a RBV specialist, specifies that the company's capacity to manage individual competencies and transform them into collective competencies is an important element of a RBV strategy. [START_REF] Edvinsson | Intellectual Capital, Realizing your company's true value by finding its hidden brainpower[END_REF] share this conception. They consider the Navigator able to measure the transformation of human capital into structural capital and the management quality concerning the flows between human and structural capital. The intellectual scorecards, through Skandia's Navigator, fulfill some of the principles of the large version of the SMA. However, the models of intellectual scorecards focuses mostly on the internal dimensions of the organization (see danish Trade Industry and Development Council, 1997). A first group of scorecards gives more importance to the human resources (this is the case of the Navigator or of Telia's strategic scorecard). A second group favours technological and informational resources (for example Carl Bro, Systemic) and a third group adopts a mixed perspective (for example the EVITA model of ABB, Celemi). These models lack the balanced design of the BSC. Also, the RBV approach has to be related to an interactive conception of the strategy. The designers of Intellectual capital Scorecards did not developed this dimension. [START_REF] Roy | Managing Intellectual Capital: the work with the Navigator in the Skandia group[END_REF] described the bottom-up approach of the Navigator through the use of an intranet network called the Dolphin system. This element is a significant evidence -but it's not enough-that the strategy is developed interactively.

In conclusion, the intellectual Scorecards are uncompleted tools of the large version of the SMA. In combining some principles of the BSC and some of the intellectual capital scorecards, it is possible to design an integrated strategic Scorecard that would fulfill the principles of the large version of SMA (see figure 3). Figure 4 shows a comparison between the Anglo-Saxon and Scandinavian strategic Scorecards.

TYPOLOGY OF THE STRATEGIC SCORECARDS AND RESULTS OF AN EMPIRICAL RESEARCH IN FRANCE THEORETICAL MODEL -CLASSIFYING STRATEGIC SCORECARDS

The typology in Figure 5 is the result of an analysis of the literature that leads to the design of Strategic Scorecards. In studying the differences of the instruments and the context within which they were elaborated, I present 6 types of strategic scorecards according to 6 variables (Table 7). These variables are based on empirical researches in management control associated to the contingency theories [START_REF] Hartmann | The appropriateness of RAPM: toward the further development of theory[END_REF].

THE FRENCH SITUATION -RESULTS

The strategic scorecards: a useful tool for managers ? -Method 1000 questionnaires were sent to companies located in France in 2001. The companies were selected using a ranking published by a major economic magazine in France (L'Expansion). Managers have first been asked on the relative importance they give to 24 generic criteria (table 2). They were also questioned on the level and accuracy of the measures by the management control tools they use (through a likert scale). The generic criteria were chosen so that the Balanced Scorecard and the Navigator would be equally represented. 112 firms have replied, 109 questionnaires were used. The variables kept are the most used generic criteria of the strategic scorecards.

Customers and competitive environment dimensions (SMA 1):

13-public image (le 2 ) 14-commercial investments (le) 15-future stakeholders' value (le)

Financial dimension (conventional management control):

5-turnover (la) 6-costs control (la) 7-shareholders' value (la)

Financial dimension (SMA version 3):

16-past stakeholders' value (la)

Human dimension : Human dimension (SMA version 3):

1 Lagging indicators. 2 Leading indicators. Table 5 gives the first answers on the relevance of the strategic scorecards. The results show the importance of the financial dimension in control management tools (turnover, costs control, shareholder value). The managers do not seem satisfied with the measure of the other dimensions. In fact, they consider important to appreciate the other dimensions within the management control tools. The average result is 4 (important); the financial and competitive dimensions are privileged (customers' satisfaction, market share, new customers, products quality and new product investments). Then, the internal dimensions (flows of informations, processes) and the human dimensions (competencies, behaviour, knowledges and know how) are mentioned. A "Societal" dimension appears to be less important (stakeholders' value, public image). Besides, some differences confirm the unsatisfaction of the managers for their management control instruments. This first analysis shows how the strategic scorecards could fulfill a gap.

Factorial analysis of the dependent variables

We have operate a transformation to the data following [START_REF] Hair | Multivariate Data Analysis[END_REF], the distribution does not disturb the interpretations. The tests of asymmetry and Kurtosis are satisfactory (-2,58<z<2,58). I proceed to a factorial analysis with principal components (PCA), confirmed by an ascending hierarchical classification on the variables (Ward's Method). The results show the significant specificity of all dependent variables and their irreductibility (using, for instance the four generic dimensions in table 2). The PCA statistics validates the distinction between the historic criteria and the forwardoriented criteria. Both types are weakly correlated. The criteria of the human dimension are the most correlated (except for individual productivity, which is quantitative). This suggest the difficulty for the managers to conceive measures for intangibles. The results indicate a relation between the human dimension, customers and the competitive environment.

Typological analysis

In order to choose between all the typological analyses methods offered by the SPSS software, I conducted a Chi-square test between the types and the dependent variables. In fact, Ward's method appears to be the most relevant. The tests were significant for all the variables except the criterium "customers satisfaction". In a second step, this criterium was excluded of the typological analysis. [START_REF] Hair | Multivariate Data Analysis[END_REF].

Besides, the small number of iterations suggests the relevance of the method. Looking at the dendogram, I observed no isolated data. I split the sample in two groups but no significant differences were found. The cluster solution is statistically representative of the general population. Table 6 characterizes each of the 6 groups. I observed 5 types of strategic scorecards and one class for companies uninterested by strategic scorecards. The first group represents the "Mixed strategic Scorecards" where the customers dimension and the internal dimension are privileged. It confirms the previous results of the factorial analysis. This type of Scorecard has been adopted, for example, by ABB, Sparbanken (Swedish Bank) and WM Data (a Swedish Consulting company) (see Mouritsen et al., 2000aMouritsen et al., , 2000bMouritsen et al., , 2000c)). The second group captures the characteristics of the BSC and the Navigator. This group is called " the Integrated Strategic Scorecard". In this group, all criteria are important. The third group defines the BSC. The fourth group concerns the companies uninterested by strategic scorecards. The fifth group is called "Structural capital Strategic Scorecard". In this group, the structural capital is dominant. This type of strategic Scorecard has been used by the Swedish Consultus company and the Danish Companies Systematic and Carl Bro. In the sixth group, the criteria of Human Capital are privileged. This group is called " the Human Capital Strategic Scorecard". The Danish PLS Consult, Ramboll corp., Telia and the Civil Aviation companies have adopted this Strategic Scorecard (Danish Trade and Industry Development Council, 1997). -the complexity of the process and the shareholder value strategy are the most significant contingency variables. -The field of activities, the instruments designers, the human competencies strategy valuation and the technological competencies strategy valuation are significant contingency variables. -1 st class: companies interested in the "mixed strategic scorecards". The financial department and the head office conceive the management control tools. The technological and informative processes of those companies are complex. They belong to four industries. (table 4). The "mixed strategic scorecards" group was not present in the "a priori" typology. It mixes aspects of the BSC (customers and competitive dimension) and some aspects of the intellectual capital strategic scorecards (through the importance of the structural capital and less importance given to shareholder value). This model doesn't seem to be associated with a specific strategic process (neither reactive nor proactive). -2 nd class: companies interested in the "integrated strategic scorecards". They are structured with very complex technological and informative processes. They belong to traditional industries (12 companies) and to the new economy (7 companies) -3 rd class: companies interested in the "BSC". They favour the shareholder value maximization and a reactive strategy (adaptation to the environment). -4 th class: companies uninterested in the strategic scorecards, mainly characterized by simple technological and informative processes. Those companies belong to the financial services industries and basic consumption goods. -5 th class: 14 companies are interested in the "structural capital strategic scorecards". They focus on project teams to conceive the management tools and a technological competencies valuation strategy.

-6 th class: 7 companies care about the "Human capital strategic scorecards". Mainly new economy companies, they favour team-projects and human competencies strategy valuation.

The statistical tests (confirmed by a Pearson correlation analysis) show that the other contingency variables do not discriminate significantly the 6 types. The type of manager (general manager, financial director, management control director, etc…) has no influence.

COMPLEMENTARY RESULTS

The material collected during the interviews I conducted confirm a significant evolution in french management accounting. Here are the four major characteristics of the evolution:

-french management accounting systems are more strategically and externally oriented (especially on competitive environment analysis), -more non financial indicators are included, -the articulation between the strategic process and the management control process remains difficult, -and the strategic scorecards and especially the BSC don't have the same success in France than in Anglo-saxon countries. When French firms use strategic scorecards, the human resources play a significant role. The chart 9 below makes a description of the firms, using a strategic scorecard, from the empirical research.

Table 9. Synthesis of interviews.

Figure 6 presents the Strategic Scorecard of Maif, a french mutual insurance company and table 10 its strategic objectives. We can see that its strategic Scorecard combines the anglosaxon and the scandinavian approaches.

CONCLUSION

This research was an opportunity to review the concepts of strategic control and strategic management accounting. I presented an instrumentation, the strategic scorecards. I proposed a generic model and a typology. I tested the interest of managers for this tool and examined the relevance of the typology. The description of the six classes demonstrated the limits of the quantitative approach. Every type of strategic scorecards deserves clarification, mostly for implementation purposes. This research is followed with a qualitative methodology investigation which is not yet completed. Some managers who answered to the questionnaire are interviewed. They have been selected for their specific profile.

Figure 2 .

 2 Figure 2. The Navigator of Skandia.

Figure 3 .

 3 Figure 3. The Integrated Strategic Scorecard.

Figure 4 .

 4 Figure 4. Comparison between anglo-saxon & scandinavian strategic scorecards.

Figure 5 .

 5 Figure 5. The determining factors of the strategic scorecards.

Figure 6 .

 6 Figure 6. Maif Strategic Scorecard.

  ABB,insurance mutualist spirit

Table 2 .

 2 Retained variables.

	Balanced Scorecard	Navigator
	Customers and competitive environment
	dimensions (SMA version 1):
	1-customers' satisfaction (la 1 )
	2-market share (la)
	3-new customers (la)
	4-products and services quality (le)

Table 3 .

 3 Companies distribution.

				complex
				processes
		Top-down			Bottom-up
		management			management
	Competitive	Stockholders	Products &	Stakeholders	Organizational	Human
	environment	& customers	processes	& processes	competencies	competencies
	TBS	Balanced	Internal	Combined	Structural capital	Human capital
	externes	Scorecards	Scorecards	Scorecards	Scorecards	scorecards
	Foreseeable environment	Uncertain environment	Turbulent environment

Table 4 .

 4 Managers offices.

	Control	Financial	General	strategy	Human	other
	managers	managers	managers	managers	resources	managers
					managers
	37	35	23	3	5	9

Table 5 .

 5 Measure and importance of the generic criteria.

	Generic criterions	average	average	differences
		measure	interest
	1-products quality (le)	2,7	4,4	1,7
	2-employees' innovation capacities (le)	2,4	3,9	1,5
	3-customers' satisfaction (la)	3	4,4	1,4
	4-part of competencies in global performances	2,6	4	1,4
	(la)
	5-public image of the company (le)	2,6	3,9	1,3
	6-employees' attitudes (le)	2,8	4,1	1,3
	7-adaptability of the processes (le)	2,9	4,1	1,2
	8-employees' knowledge & know-how (le)	2,8	4	1,2
	9-effectiveness of the processes (la)	2,9	4	1,1
	10-control of the information flows (le)	3,2	4,2	1
	11-effectiveness of the infrastructures (la)	2,9	3,8	0,9
	12-commercial investments (le)	3,3	4,1	0,8
	13-adaptability of the infrastructures (le)	2,9	3,7	0,8
	14-employees' productivity (la)	3,2	3,8	0,6
	15-employees' satisfaction (la)	3,1	3,7	0,6
	16-market shares (la)	3,5	4	0,5
	17-acquisition of new customers (la)	3,6	4,1	0,5
	18-investments in research & development (le)	3,2	3,7	0,5
	19-past stakeholders' value (le)	2,8	3,3	0,5
	20-future stakeholders' value (le)	2,8	3,1	0,3
	21-training investments (le)	3,5	3,8	0,3
	22-costs management (la)	4	4,1	0,1
	23-shareholders' value (la)	4	4	0
	24-turnover (la)	4,8	4	0,8 (-)

Table 6 .

 6 Characteristics of the six categories discriminated.Description of the companies' classesI have tried to verify, using a discriminant analysis, if some contingency variables (independent variables) differ significantly from one class to another. The Pearson Chi-square tests proposed in table 7 show that:

	Variables retained	group 1 group 2 group 3 group 4 group 5 group 6
	2-market share	4,6	4,3	3,6	3,5	4,4	3,2
	3-new customers	4,6	4,5	4,1	3,5	3,6	4,5
	4-products quality	4,4	4,8	4,7	4,0	3,9	4,7
	5-turnover	4,3	4,4	4,0	3,7	4,2	4,2
	6-costs management	3,9	4,4	4,5	3,6	4,9	3,3
	7-shareholders' value	3,3	4,5	4,8	3,9	3,8	3,9
	8-individual productivity	3,7	4,0	4,5	3,0	3,5	3,7
	9-employees' satisfaction	3,5	4,5	3,7	3,0	3,2	3,7
	10-processes effectiveness	4,1	4,6	4,4	3,3	4,0	3,5
	11-infrastructures effectiveness	4,0	4,1	3,9	2,9	4,3	3,8
	12-research & development	3,5	4,6	3,3	2,9	4,5	4,3
	13-public image	3,8	4,4	3,7	3,6	3,7	4,5
	14-comercial investments	4,1	4,7	4,1	3,5	4,2	3,9
	15-future stakekeholders' value	3,1	3,9	3,0	2,4	3,2	3,6
	16-past stakeholder's value	3,2	4,2	3,8	2,4	3,1	2,9
	17-competencies value	4,4	4,5	4,0	3,1	3,2	4,5
	18-knowledge and know-how	3,9	4,4	4,0	3,3	3,0	5,0
	19-strategic training	3,9	4,4	3,9	3,0	3,2	4,7
	20-behaviour	4,1	4,7	4,2	3,2	3,6	4,8
	21-innovative capacities	3,9	4,6	3,6	2,9	3,9	4,7
	22-processes adaptation	3,3	4,0	3,9	3,0	4,5	4,7
	23-infrastructures adaptation	3,7	4,6	4,4	3,0	4,7	4,7
	24-information flows	4,3	4,6	4,5	3,1	4,4	4,8

Table 7 .

 7 Contingency Variables.

	Independant variables	Chi-square Signification
	Activity fields	2,724	0,024
	Complexity of the processes	3,456	0,007
	Nature of the structure of the firms	1,864	0,110
	Tools devisers	3,068	0,013
	Environment uncertainty	0,690	0,632
	Strategy of human ressources' valuation	2,455	0,039
	Strategy of adaptation to environmental evolutions	1,090	0,371
	Strategy of satisfaction of the stakeholders	1,885	0,105
	Strategy of valuation of the technological competencies	2,740	0,024
	Strategy of optimization of the processes	0,059	0,448
	Strategy of max. of the shareholders' value	4,273	0,002

Table 8

 8 below describes the characteristics of the 6 classes.

Table 8

 8 gives a synthesis of the typological analysis.

	Types	size	favoured criterions	Companies
				characteristics
		24 companies	qualitative criteria	complex processes,
	Combined Strategic		concerning first	general & financial
	Scorecards		customers &	management for the
			organization	conception
	Integrated Strategic	17 companies	the 24 criteria are	technological &
	Scorecards		considered as important	informative processes
				are very complex,
				traditional & new
				economy favoured
	Balanced Scorecard	34 companies	first, financial	strategy of shareholder
			dimension, then	value growth, new
			customer dimension	economy & traditional
				industry both
	Strategic Scorecards	13 companies	financial criteria only simple processes, banks,
	rejected			assurance, ordinary
				consumption goods
	Structural Capital	14 companies first, qualitative criteria	project teams for the
	Strategic Scorecards		concerning organization	conception,
			(processes, technology..)	technological
				competencies valuation
	Human Capital	7 companies first, qualitative criteria	new-economy, project
	Strategic Scorecards		concerning human	teams, human
			capital	competencies valuation

Table 10 .

 10 Maif strategic objectives.

	Economic & Mutualist Perspective:	Internal Processes Perspective:
	1-To promote the mutual insurance system,	11-Effective organizational processes,
	2-To reach objectives of profitability,	12-Innovative & learning organization,
	3-To develop activities of prevention, &	13-Adaptable processes,
	sponsoring,..	14-Costs control,
	4-To increase commercial margins,	15-To develop performant management
	5-To develop subsidiaries	instruments
	Members of the Mutual Perspective:	Organizational Learning Perspective:
	6-To develop members' loyalty,	16-Employees' satisfaction & loyalty,
	7-To increase market shares,	17-Performant information systems,
	8-Support for our values,	18-Employees' motivation,
	9-Partnership develpment,	19-To increase in-service training,
	10-To develop appropriate services	20-To develop employees' competencies

The three

Types of Characteristics Some versions research authors of the SMA


Dr Andreas Ohlin
email: andreas.ohlin@orebroll.se

Anders Bäckman

Bo Söderquist

; Sten

Maria Björkqvist

Richard V Goering

Sten Wingren

Rapid typing of neonatal Staphylococcus epidermidis isolates using PCR for repeat regions in surface protein genes

Keywords: 1, Staphylococcus epidermidis 2, genotype 3, PCR 4, human Staphylococcus epidermidis, genotype, PCR, human

Purpose. Staphylococcus epidermidis is a significant pathogen in neonatal sepsis and other nosocomial infections. For further investigations of the colonisation patterns and invasive pathways typing methods that are applicable on large populations of bacterial isolates are warranted. Methods. In the present study a genotyping method based on polymerase chain reaction (PCR) for the repeat regions of four genes (sdrG, sdrF, aap and sesE ) that encode for bacterial surface proteins was developed and applied to a sample of well-characterised neonatal blood isolates of S. epidermidis (n = 49). The PCR products were visualised on agarose gel (sdrG, sdrF and ses E) or by fragment analysis (aap). The discriminatory index (D-index) for genotyping of the different genes was compared to genotyping by pulsed-field gel electrophoresis.

Results. The highest D-index for the PCR-based typing methods was found for the combination of sdrF, sdrG, and aap (D-index 0.94), whereas the optimal two-gene combination (sdrF and aap) resulted in a D-index of 0.92. Conclusions. We conclude that the described method can be used for genotyping of large populations of S. epidermidis isolates with a sufficient discriminatory capacity, and we suggest that the combination of sdrF and aap is most suitable to use.

Introduction

Neonatal sepsis is a common complication in neonatal intensive care. Premature birth, low birth weight, and long hospital stay are significant risk factors for this condition [START_REF] Healy | Features of invasive staphylococcal disease in neonates[END_REF]. In parallel with the survival of more premature and vulnerable infants, S. epidermidis, which is known as a commensal, has emerged as the most common cause of neonatal sepsis [START_REF] Bizzarro | Seventy-five years of neonatal sepsis at Yale: 1928-2003[END_REF]. These infections are often caused by multiresistant strains that can persist in neonatal intensive care units (NICUs) for long periods [START_REF] Klingenberg | Persistent strains of coagulase-negative staphylococci in a neonatal intensive care unit: virulence factors and invasiveness[END_REF][START_REF] Bjorkqvist | Phenotypic and genotypic characterisation of blood isolates of coagulase-negative staphylococci in the newborn[END_REF][START_REF] Vermont | Persistence of clones of coagulase-negative staphylococci among premature neonates in neonatal intensive care units: two-center study of bacterial genotyping and patient risk factors[END_REF][START_REF] Villari | Molecular epidemiology of Staphylococcus epidermidis in a neonatal intensive care unit over a three-year period[END_REF][START_REF] Hira | Clinical and molecular epidemiologic characteristics of coagulase-negative staphylococcal bloodstream infections in intensive care neonates[END_REF][START_REF] Widerstrom | Molecular epidemiology of meticillin-resistant coagulase-negative staphylococci in a Swedish county hospital: evidence of intra-and interhospital clonal spread[END_REF][START_REF] Monsen | Spread of clones of multidrug-resistant, coagulase-negative staphylococci within a university hospital[END_REF].

To prevent the spreading of these strains, additional knowledge of the factors that influence the colonisation of newborns, the invasive routes, and other pathogenetic factors of S. epidermidis is warranted. These issues cannot be investigated without manageable typing methods requiring an acceptable workload that can fingerprint the large numbers of S. epidermidis isolates.

In the search for typing tools applicable to large samples of bacterial isolates, sequencing of the repeat regions in the genes encoding S. epidermidis surface proteins, such as aap and sdrG, has been suggested as a novel typing method [START_REF] Monk | Use of outer surface protein repeat regions for improved genotyping of Staphylococcus epidermidis[END_REF]. The aim of the present study was to develop a method that combines the diversity in the serine-aspartate dipeptide repeats with an easy-to-use detection approach, and to apply this method to a well-characterised collection of neonatal blood isolates, thus enabling comparison with pulsed-field gel electrophoresis (PFGE).

Materials and Methods

Bacterial isolates

Fifty clinical isolates of S. epidermidis previously determined to species level by Staph-zym (Rosco Diagnostica, Taastrup, Denmark), obtained from blood cultures of neonates and collected from 1990 to 1997 at the Neonatal Intensive Care Unit, Örebro University Hospital, were included in this study. After subcultivation, one of the bacterial isolates was identified as a S. hominis by sequencing of the rpoB gene [START_REF] Hellmark | Simultaneous species identification and detection of rifampicin resistance in staphylococci by sequencing of the rpoB gene[END_REF] and was hence excluded, leaving the remaining 49 S. epidermidis isolates for further analysis. These isolates have previously been characterised phenotypically and genotypically, including PFGE [START_REF] Bjorkqvist | Phenotypic and genotypic characterisation of blood isolates of coagulase-negative staphylococci in the newborn[END_REF]. According to the clinical and laboratory criteria applied in the former study, 11 isolates were classified as representing true neonatal sepsis and 38 isolates were considered as contaminants. All isolates were stored at -70˚C pending analysis and were subcultured on blood agar plates (4.25% Columbia II Agar, BBL, Becton Dickinson, MD, USA; 0.3% Agar No. 2, Lab M Ltd., Bury, UK; 5% bovine blood) and checked for purity prior to DNA preparation.

Pulsed-field gel electrophoresis

The isolates were characterised by PFGE of chromosomal SmaI digests, prepared with the GenePath Group 1 Reagent Kit (Bio-Rad Laboratories, Hercules, CA, USA), and these data originated from a previous study [START_REF] Bjorkqvist | Phenotypic and genotypic characterisation of blood isolates of coagulase-negative staphylococci in the newborn[END_REF].

DNA preparation

A QIAamp DNA Mini Kit was used to isolate DNA from the strains (Protocol D, QIAGEN, Hilden, Germany) with the following modifications; Bacteria obtained from the blood agar plate were dissolved in sterile saline and pelleted, dissolved in 180 μL ATL buffer (QIAGEN) with10 μL mutanolysin 10 U/μL (Sigma, St. Louis, MO, USA) added and incubated at 37˚C for 30 min. The mix was treated with proteinase K and AL buffer (QIAGEN) at 56˚C for 30 min and DNA-purified according to the tissue protocol, using ethanol and wash buffers supplied in the kit, and finally eluted in 50 μL AE buffer (QIAGEN) and stored at 4˚C.

PCR

The repeat regions for aap and sdrG were amplified by PCR, as previously described by Monk and Archer [START_REF] Monk | Use of outer surface protein repeat regions for improved genotyping of Staphylococcus epidermidis[END_REF], using Taq PCR Master Mix Kit (QIAGEN, Hilden, Germany) (25 μL/50 μL reaction), 2 μM of each primer (Applied Biosystems, Warrington, UK) and 2 μL DNA preparation. The primers for sdrF and sesE (Scandinavian Gene Synthesis AB, Köping, Sweden) were designed using the PRIMER3 program and compared with published sequences on BLAST (Table 1). To facilitate fragment analysis of the products one primer in each pair was purchased labelled with VIC and 6-FAM, respectively.

The sdrF, sdrG, and sesE amplicons (6 μL) were treated with restriction enzymes for 2 hours.

SdrG was treated with HpyCH4III (New England Biolabs Inc. (NEB), Ipswich, MA, USA) 5 U at 37˚C, sdrF with BtsCI (NEB) 10 U at 50˚C, and sesE with BstYI (NEB) 5 U at 60˚C, respectively, and aap was analysed without any enzyme treatment. Enzymes were selected using the NEB cutter computer program.

The PCR products and restriction fragments were separated and visualised on a 1.5% agarose gel (Agarose 1000, 1 × TBE, 100 V, 60 min) stained with ethidium bromide. The stained bands were compared to a size marker on each gel (100 bp DNA ladder, Gibco BRL, Carlsbad, CA, USA).

The product sizes of all aap PCR products and the enzyme-treated fragments from sdrF, sdrG, and sesE that yielded similar visual results were confirmed by fragment analysis, using ABI 310 or ABI 3130xl, and compared to LIZ-1200 (GeneScan 1200 LIZ, ABI, Foster City, CA, USA) size standard in Peakscanner 1.0/GeneMapper 4.0 (ABI).

Statistical methods

To evaluate discriminatory capacity of different PCR methods, a discrimination index (D-index) for each included gene and for combinations of the studied genes was calculated [START_REF] Hunter | Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity[END_REF]. Briefly, the D-index represents the likelihood that two randomly picked strains from the sample are separated by the method and recognised as two different strains. This means that a high D-index (close to one) divides the sample into many small groups, whereas, a low D-index (close to zero) indicates that the sample is only divided into a few large groups.

Confidence intervals of 95% were calculated, as described by Grundmann et al. [START_REF] Grundmann | Determining confidence intervals when measuring genetic diversity and the discriminatory abilities of typing methods for microorganisms[END_REF].

Results

The sdrG, sdrF, and sesE repeats were present in all 49 isolates, whereas aap was detected in 38/49 isolates. When the primary PCR products of sdrG, sdrF, and sesE were visualised on the agarose gels, all genes had a single predominating genotype, sdrG, 35 isolates; sdrF, 36 isolates; and sesE, 44 isolates, respectively, with amplicons so similar in size that they were indistinguishable on the gel. To further enhance the discrimination between these genotypes, the PCR products were treated with restriction enzymes, and the subtypes of isolates with similar band patterns were confirmed by fragment analysis.

For aap the primary PCR products of all bacterial isolates that carried the aap gene were of similar size and thus difficult to separate on the agarose gel. Since no suitable restriction enzyme for the product could be identified, the sizes of these amplicons were instead determined by fragment analysis. sdrG After treatment with HpyCH4III restriction enzyme, the sdrG samples showed 2-4 distinct bands of a size ranging from 100 to 300 base pairs (bp), representing 13 different genotypes (Table 2). The most common genotype included 25 isolates, which all had identical band patterns. Four genotypes comprised 6, 5, 3, and 2 isolates, respectively, and the final 8 genotypes were only represented by single isolates. sdrF After treatment with BtsCI restriction enzyme, the sdrF samples showed 1-3 clear distinct bands in the size range of 250-1,000 bp that were used for typing and corresponded to 17 different genotypes. The most common genotype consisted of 19 isolates with identical band patterns (Table 2). Five genotypes included 7, 5, 3, 3, and 2 isolates, respectively, and 11 genotypes were only represented by single isolates.

sesE

After treatment with BstYI restriction enzyme, the sesE samples showed 1-5 distinct bands in the size range of 100-700 bp. These were used for typing and resulted in 8 different genotypes (Table 2). Twenty-four isolates constituted the most common genotype, and the others had 12, 4, 3, and 3 strains, respectively. Three genotypes were represented by single isolates.

aap Since the aap repeat was found in 38/49 isolates, one genotype consisted of 11 isolates without aap. The remaining 38 isolates where analysed with fragment analysis showing a size range of 414-514 bp and 7 genotypes with 13, 11, 8, 2, 2, 1, and 1 strains respectively.

PFGE

Six isolates were indistinguishable by PFGE, and 17 were classified as single isolates. The remaining isolates were assigned to various genotypes with 2 or 3 isolates. The 6 indistinguishable isolates were also indistinguishable when analysing sdrG, aap and sesE.

Regarding sdrF, 5 isolates showed an identical result, whereas, one isolate had a different genotype. The 17 single isolates were subdivided into 11 groups with sdrG and sdrF, 7 groups with aap, and 6 groups with sesE.

Discriminatory index

All the calculated D-indices are presented in Table 2. The highest D-index for a single gene was found for sdrF (0.82), followed by aap, sdrG, and sesE, respectively. By combining two genes, the highest D-index was found in aap/sdrF (0.92), followed by aap/sdrG. When a third gene was added, the D-index was only marginally enhanced (aap/sdrG/sdrF, D-index 0.94).

The corresponding figure for PFGE was 0.98.

Correlation between PFGE and surface protein PCR results

When comparing results of PFGE and the PCR genotyping method, we found concordance in the vast majority of isolates. According to PFGE three clusters including 9, 5 and 5 closely related isolates respectively were identified; the remaining 30 were divided into 5 pairs and 20 single isolates. Using all four of the investigated genes the repeat region PCR characterized the three clusters in exactly the same way as PFGE with the exception of 2 isolates in the largest cluster, where the result for sdrF diverted. One of the clusters including five isolates showed exactly the same genotypes as 2 other isolates that were grouped into one pair and one single isolate according to PFGE.

Discussion

In this study we present a novel typing method for S. epidermidis based on PCR and the use of restriction enzymes. The method combines feasibility and low cost with a high discriminatory capacity. We used a gel-based detection in combination with fragment analysis to evaluate the replicates, which enables the method to be applicable to large collections of samples.

S. epidermidis constitutes the predominant bacterial species of the normal skin flora and is represented by numerous isolates per individual but is also a significant nosocomial pathogen in predisposed patients. Nosocomial strains of S. epidermidis regularly display multiple antibiotic resistance, but the emergence of resistance also to glycopeptides during the recent years further impose the importance of epidemiological surveillance [START_REF] Biavasco | Glycopeptide resistance in coagulasenegative staphylococci[END_REF][START_REF] Center | Decreased vancomycin susceptibility of coagulase-negative staphylococci in a neonatal intensive care unit: evidence of spread of Staphylococcus warneri[END_REF]. Thus there is an urgent demand for swift, easy-to-use typing methods for S. epidermidis not only for research purposes but also for clinical use, for instance in case of an out-break of multi-resistant bacterial strains in a hospital setting Its our opinion that PCR of repeat regions can become such a tool that can be applied in a situation when an epidemiological survey has collected hundreds of isolates, and there is need to quickly and easily trim down the number of interesting isolates that qualify for further analysis.

In 2007 Monk and Archer [START_REF] Monk | Use of outer surface protein repeat regions for improved genotyping of Staphylococcus epidermidis[END_REF] suggested sequencing of repeat regions in aap and sdrG as a typing system for S. epidermidis and showed that there was sufficient variation in these genes to make them suitable candidates for gene-based typing. Sequencing of the repeat regions gives detailed information and facilitates interlaboratory comparisons, but on the other hand, it is complicated, expensive, and time-consuming, which limits its use to small samples. Therefore we instead suggest a gel-based approach to minimize costs and, thus, make the method more suitable for epidemiological studies. Presumably because of these protocol differences, our D-indices for aap and sdrG seem to be lower than those published by Monk and Archer. To counteract this loss of information when sequencing is replaced by a gel-based approach, two additional genes were studied, and by combining these genes, we can now present a gel-based typing method with a discriminatory power that is comparable to the sequence-based method that Monk and Archer described.

In the present study PFGE was used as a reference method. PFGE or multilocus sequence typing (MLST) are widely used for detailed genotyping of S. epidermidis, but are too laborious to be applicable to large samples of bacterial strains [START_REF] Klingenberg | Persistent strains of coagulase-negative staphylococci in a neonatal intensive care unit: virulence factors and invasiveness[END_REF][START_REF] Villari | Molecular epidemiology of Staphylococcus epidermidis in a neonatal intensive care unit over a three-year period[END_REF][START_REF] Monk | Use of outer surface protein repeat regions for improved genotyping of Staphylococcus epidermidis[END_REF][START_REF] Sivadon | Polymorphism of the cell wall-anchoring domain of the autolysin-adhesin AtlE and its relationship to sequence type, as revealed by multilocus sequence typing of invasive and commensal Staphylococcus epidermidis strains[END_REF][START_REF] Thomas | Improved multilocus sequence typing scheme for Staphylococcus epidermidis[END_REF] . The high correlation between PFGE and repeat region PCR found in this study might indicate that the methods share a common biological background. On the basis of our data we suggest that the PCR-based typing method presented in this article can be used as an alternative to PFGE or MLST, when high speed and low costs are a higher priority than maximum discriminatory capacity.

We targeted repeat regions in four genes encoding different cell-wall-anchored proteins.

These proteins have been suggested as important factors in the attachment and interaction between bacteria and human cells. There are additional data supporting the view that some of these proteins are expressed during clinical infections with S. epidermidis [START_REF] Bowden | Identification and preliminary characterization of cell-wall-anchored proteins of Staphylococcus epidermidis[END_REF][START_REF] Mccrea | The serine-aspartate repeat (Sdr) protein family in Staphylococcus epidermidis[END_REF]. It has been suggested that repeats are a way to form coil-like structures that enable the protein to span the cell wall (SD-repeats), or to project the A region of the protein away from the cell wall [START_REF] Bowden | Identification and preliminary characterization of cell-wall-anchored proteins of Staphylococcus epidermidis[END_REF].

Our choice of genes was based on the fact that they had repeat regions with a high rate of variation, as well as a proven stability when recultured [START_REF] Monk | Use of outer surface protein repeat regions for improved genotyping of Staphylococcus epidermidis[END_REF]. The genes have also been reported to have a high prevalence in sepsis strains, contaminants and skin flora [START_REF] Bowden | Identification and preliminary characterization of cell-wall-anchored proteins of Staphylococcus epidermidis[END_REF][START_REF] Hartford | The Fbe (SdrG) protein of Staphylococcus epidermidis HB promotes bacterial adherence to fibrinogen[END_REF][START_REF] Nilsson | A fibrinogenbinding protein of Staphylococcus epidermidis[END_REF]. In our material 78% of the isolates were positive for aap and 100% for the other genes.

We evaluated four different genes and, as presented in Table 2, no single repeat analysis had a satisfactory discriminatory power. Therefore, we also studied combinations of genes and, in our opinion, two genes were sufficient in this sample to achieve what we consider to be an acceptable discriminatory capacity. Adding a third gene only marginally increased the discriminatory capacity. We, therefore, suggest the combination of aap and sdrF as suitable for genotypic typing of neonatal S. epidermidis isolates, but before the generalisation of these results can be stated, other series of S. epidermidis strains with a more heterogeneous origin should evaluated.

Conclusion

We present a novel gel-based typing method for S. epidermidis based on PCR of four genes encoding cell-wall proteins and the use of restriction enzymes. The method combines feasibility and low cost with a high discriminatory capacity, comparable to that of PFGE and sequence-based methods. In our study a combination of two genes, aap and sdrF, was found to be optimal, but other combinations of the four studied genes could be more suitable when applied to another material of S. epidermidis isolates. Our proposed method could be used for epidemiological investigations, for example in case of outbreaks of multiresistant strains or exploring the dynamics of the colonization pattern of newborns. In addition, it enables pathogenic studies of, for example, tracking invasive routes of clinical isolates as well as identification/recognition of virulent clones.

Table 1.

Details of the 4 PCR methods used to study the 49 S. epidermidis isolates in this study.

Gene

  -CTGATGGGGAAGATGTTCGT-3´) SDRF2 (5-´TCATGATCTTCATTTGCTCCTG-3´) SE1L (5´-CATCTTTATCTGTACCGCCTGT-3´) SE1R (5´-ATCCAACTGCCACTGAACCT-3´).repeat region following the A (ligand) region Serine-aspartate repeatrepeat region consisting of serine and aspartate that is located downstram from the B-region. Nomenclature adapted from Bowden et al.[START_REF] Bowden | Identification and preliminary characterization of cell-wall-anchored proteins of Staphylococcus epidermidis[END_REF]

Table 1 and 2

 12

Table 2 .

 2 Number of genotypes, and discrimination index (D-index) for the included repeats and combinations of repeats in the included S. epidermidis neonatal blood isolates (n = 49).

	Gene	Number of	No. of isolates with	D-index	95% confidence
		genotypes	most common		interval
			genotype (%)
	aap	8	13 (27)	0.81	0.77-0.86
	sdrG	13	25 (51)	0.72	0.59-0.85
	sdrF	17	19 (39)	0.82	0.73-0.92
	sesE	8	24 (49)	0.70	0.59-0.80
	aap sdrG	18	13 (27)	0.89	0.84-0.94
	aap sdrF	21	11 (22)	0.92	0.87-0.96
	aap sesE	16	11 (22)	0.88	0.83-0.93
	sdrG sdrF	20	15 (31)	0.88	0.81-0.95
	sdrG sesE	17	21 (43)	0.80	0.69-0.91
	sdrF sesE	22	15 (31)	0.88	0.81-0.95
	aap sdrG sdrF	23	8 (16)	0.94	0.91-0.97
	aap sdrG sesE	21	11 (22)	0.91	0.87-0.96
	aap sdrF sesE	24	11 (22)	0.92	0.88-0.97
	sdrG sdrF sesE	22	12 (24)	0.91	0.86-0.96
	PFGE	34	6 (12)	0.98	0.96-0.99

Acknowledgements

We would like to thank Anders Magnusson for statistical support and Bengt Hellmark for rpoB sequencing. This work was supported by Örebro University and the Research Committee of Örebro University Hospital.


Michael Wong

John M Woodley

Gary J Lye
email: g.lye@ucl.ac.uk

Á G J Lye

Application of bipolar electrodialysis to E. coli fermentation for simultaneous acetate removal and pH control

Keywords: Acetate removal, Bipolar electrodialysis, E. coli TG1, pH control

The application of bipolar electrodialysis (BPED) for the simultaneous removal of inhibitory acetate and pH control during E. coli fermentation was investigated. A two cell pair electrodialysis module, consisting of cation exchange, anion exchange and bipolar membranes with working area of 100 cm 2 each, was integrated with a standard 7 l stirred tank bioreactor. Results showed that BPED was beneficial in terms of in situ removal of inhibitory acetate and a reduction in the amount NH 4 OH used for pH control. In batch and fed-batch BPED fermentations, base additions were decreased by up to 50% in both cases compared to electrodialysis (ED) fermentations with pH controlled at 6.7 ± 0.1. Consequently, the final biomass (34.2 g DCW l -1 ) and recombinant protein (5.5 g l -1 ) concentrations obtained were increased by up to 37 and 20%, respectively.

Introduction

E. coli is widely used for the production of recombinant enzymes and therapeutic proteins. One of the factors limiting the productivity of E. coli fermentations is the formation of acetate which causes inhibition of cell growth and recombinant protein synthesis [START_REF] Han | Acetic acid formation in Escherichia coli fermentation[END_REF]. A number of approaches exist to overcome this problem [START_REF] Eiteman | Overcoming acetate in Escherichia coli recombinant protein fermentations[END_REF], the most widely used being controlled feeding of the carbon source [START_REF] Akesson | Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding[END_REF]. Recently we have demonstrated the benefits of electrodialysis (ED) applied to E. coli fermentation to reduce the concentration of acetate in the media and hence increase both cell growth and the specific recombinant protein production [START_REF] Wong | Enhanced recombinant protein expression in batch and fed-batch Escherichia coli fermentation based on removal of inhibitory acetate by electrodialysis[END_REF]. Concerns remain however regarding the volume of NH 4 OH added for pH control which could also be toxic to the cells [START_REF] Muller | Ammonium toxicity in bacteria[END_REF]. A potential solution is the use of bipolar electrodialysis (BPED) which can hydrolyse an aqueous salt solution under the influence of electrical current to produce H 2 and OH -. In principle this approach could be used to directly acidify or basify the fermentation broth without the addition of extraneous chemicals. This technology has been widely used in the production of organic acids [START_REF] Huang | Application of electrodialysis to the production of organic acids: stateof-the-art and recent developments[END_REF][START_REF] Couallier | Three-compartment bipolar membrane electrodialysis for splitting of sodium formate into formic acid and sodium hydroxide: role of diffusion of molecular acid[END_REF], amino acids [START_REF] Grib | Extraction of amphoteric amino acids by an electromembrane process. pH and electrical state control by electrodialysis with bipolar membranes[END_REF] and in the field of environmental protection [START_REF] Koter | Electromembrane processes in environment protection[END_REF]. From an operational perspective, the cost of acid or base could be reduced and the process could have reduced requirements with regard to treatment of the spent broth.

The aim of this work is therefore to demonstrate the principle of BPED fermentations and compare cell growth and protein production to our previous studies on batch and fed-batch fermentations with or without ED application [START_REF] Wong | Enhanced recombinant protein expression in batch and fed-batch Escherichia coli fermentation based on removal of inhibitory acetate by electrodialysis[END_REF]). To the best of our knowledge no previous study has reported simultaneous acetate removal and pH control of E. coli fermentation by BPED.

Materials and methods

The E. coli strain, culture media, equipment and operating conditions used for shake-flask, batch and fed batch fermentations were the same as described previously [START_REF] Wong | Enhanced recombinant protein expression in batch and fed-batch Escherichia coli fermentation based on removal of inhibitory acetate by electrodialysis[END_REF], as were all the analytical protocols. All cultures of E. coli TG1 pGLO were induced at early exponential phase with 0.25% (w/v) L-arabinose for the synthesis of recombinant green fluorescent protein (GFP). Results represent typical profiles from at least duplicate independent fermentations.

Bipolar electrodialysis module and fermentations

Figure 1 shows a schematic diagram of the BPED system used. The module consists of six equal volume compartments fitted with two electrodes at each end, supported by one-cation exchange, two-anion exchange and two-bipolar membranes (Neosepta, Tokuyama Corporation, Japan) having a total effective membrane area of 200 cm 2 . Prior to operation, the module was chemically sterilised with 1 M NaOH. When BPED was applied, the fermentation broth was circulated through compartment F at 500 ml min -1 , while 2 l of 2 M Na 2 SO 4 flowed through compartments A, R and C at the same rate. Automatic pH control was achieved using a proportional-integral-derivative (PID) controller, linking measured pH with applied current. When the pH fell below the set point of 6.7, due to the production of acetic acid, the power output was increased accordingly, up to a maximum current of 5 A and a maximum voltage of 40 V. This resulted in the generation of OH -from the bipolar membrane in compartment F as well as removal of acetate ions from the fermentation broth in compartment F to compartment R via the anion exchange membrane. When the pH returned to pH 6.7, the current was switched off, so the pH remained constant, such that there would not be any removal of ions from compartment F.

The use of BPED thus allows pH control without direct acid or base addition. In the media formulation used, some NH 4 OH addition is required as a nitrogen source [START_REF] Korz | Simple fed-batch technique for high cell density cultivation of Escherichia coli[END_REF]. In order to facilitate this, the dynamic of the PID controller used for BPED unit operation was reduced by 20%, so that the rate of hydroxyl ions produced was decreased. Consequently, some NH 4 OH was added by the conventional pH control system of the fermenter but at a rate more closely linked to the nitrogen requirements of the cells rather than that required for pH control.

Results and discussion

Ammonium toxicity

To demonstrate the impact of ammonium toxicity on the cells, a small scale preliminary experiment was performed involving additions of 10, 20 and 30 g NH 4 Cl l -1 to separate 100 ml shake-flask cultivations. These amounts were indicative of the quantity of ammonium used for pH control in our previous batch and fed-batch fermentations [START_REF] Wong | Enhanced recombinant protein expression in batch and fed-batch Escherichia coli fermentation based on removal of inhibitory acetate by electrodialysis[END_REF]. Figure 2 gives an indication of ammonium toxicity on growth of the E. coli TG1 cells. Once NH 4 Cl was added at mid-exponential phase, there was an immediate effect on the cells resulting in a reduction of cell growth rate. Addition of 30 g NH 4 Cl l -1 led to a 50% reduction in the final biomass concentration. These data suggest that the ammonium concentrations in the broth should be maintained below 10 g l -1 .

Batch and fed-batch BPED fermentations

The results of batch and fed-batch BPED fermentations are presented in Figs. 3 and4, respectively. Reproducible trends for cell growth, acetate accumulation and recombinant protein production were obtained in replicate independent experiments. In batch BPED fermentations, BPED was applied after the lag phase at 6 h and arabinose was added to induce GFP synthesis at early exponential phase. The biomass and protein concentrations continued to increase to 15.4 and 3.3 g l -1 respectively at 18 h. Similar trends can be observed for the initial phase of the fed-batch BPED fermentation. When exponential feeding started at 10 h, biomass and protein concentrations increased linearly up to 30 h, reaching final concentrations of 34.2 and 5.5 g l -1 respectively. Figures 3b and4b indicate the corresponding base additions and power output of each experiment. These are important parameters of BPED performance and are discussed further in the next section.

Comparison of ED and BPED fermentations

Table 1 summarises the results of the BPED fermentations performed here and compares them to our previous results from batch and fed-batch fermentations with or without application of conventional electrodialysis. The presence of acetic acid retards cell growth and inhibits recombinant protein production [START_REF] Han | Acetic acid formation in Escherichia coli fermentation[END_REF]). Consequently we were able to show that the application of conventional ED for in situ acetate removal enhanced cell growth and recombinant protein synthesis [START_REF] Wong | Enhanced recombinant protein expression in batch and fed-batch Escherichia coli fermentation based on removal of inhibitory acetate by electrodialysis[END_REF]. A limitation of the earlier ED studies was that the anion exchange membrane area was limited to 100 cm 2 . It is evident in this work that by doubling the anion exchange membrane area to 200 cm 2 , the rate of acetate ion removal is increased and so the In addition to removal of acetate, however, ED also removed OH -from compartment F to R and as much as 50% extra base addition was required for pH control. In contrast to the ED fermentations, the additional PID controller used in the BPED system led to the rate of ion removal being proportional to the amount of acetate present so fewer OH -were removed. This conservation of OH -in compartment F and simultaneous generation of OH -for pH control from the bipolar membrane resulted in reduction of base addition by up to 60% in batch and 71% in fedbatch mode respectively. In addition, by providing a less toxic environment for cell growth and further reductions in acetate accumulation, there was an increase in biomass yield of up to 37% in fed-batch fermentation and an increase in recombinant protein production of up to 20%.

With respect to operation of the ED/BPED units reduced energy consumption is important as it leads to a reduction in operating cost and reduced environmental footprint of the process. ED fermentations operated at a fixed current of 2 A with a voltage range of 35-40 V, so the power output was fixed at 70-80 W. From Figs. 3b and 4b, the calculated average power output for BPED batch and BPED fed-batch fermentations were 6.5 and 59 W, respectively. Based on the ED power consumption data there appears to be a specific power consumption of the order of 10 kW m -3 . However, this is due to the small scale and experimental nature of the ED device used. The lower energy consumption is a major advantage of BPED over ED. Furthermore, due to the lower power output with BPED, electromigration only took place when acetate was present, so energy was not wasted in the removal of other ions. This is even more significant at industrial scale where energy consumption and base used can be major cost factors.

Conclusions

This work has demonstrated for the first time the use of a PID controller system for bioreactor pH control and simultaneous in situ removal of inhibitory acetate by bipolar electrodialysis. BPED was able to remove up to 75% of acetate formed from the fermentation broth. Furthermore, it served to provide base pH control and therefore the total volume of base addition was decreased by 50%. Consequently, there were increases in biomass yield of up to 37% in fedbatch fermentation and in recombinant protein production of up to 21%. We conclude that this approach could therefore be applied beneficially to a wide range of E. coli fermentations for production of recombinant enzymes and therapeutic proteins.

Fig. 1

 1 Fig. 1 Principle of bipolar membrane electrodialysis for simultaneous in situ removal of acetic acid from E. coli fermentation broth and bioreactor pH control: AEM anion-exchange membrane; BPM bipolar membrane; A anode compartment; R recovery compartment; F fermentation compartment; C cathode compartment

Fig. 2

 2 Fig. 2 Effect of ammonia on growth of E. coli TG1 pGLO in shake-flask fermentations with additions of 0 (open circle), 10 (filled square), 20 (filled triangle) and 30 (filled circle) g NH 4 Cl l -1 . Arrow indicates time of ammonium chloride addition

Fig. 3

 3 Fig. 3 Bipolar electrodialysis batch fermentation kinetics of E. coli TG1 pGLO. a Off-line data for biomass (filled circle), acetate (filled square) and recombinant protein, GFP (open triangle) concentration. b Power output (dash) and base addition (filled square). Arrows indicate times of induction and BPED application

Table 1

 1 Comparison of batch and fed-batch E. coli TG1 pGLO fermentation kinetics with standard electrodialysis (ED) application and application of bipolar electrodialysis (BPED)

	Fermentation	[Biomass]	[Protein]	[Acetate]	Volume of base	References
	conditions	(g DCW l -1 )	(g l -1 )	(g l -1 )	addition (ml)
	Batch	13.6	2.9	5.5	100	Wong et al. (2009)
	ED batch	12.3	3.1	2.1	125	Wong et al. (2009)
	BPED batch	15.4	3.3	1.3	50	This work
	Fed-batch	30.5	5.0	25.1	800	Wong et al. (2009)
	ED fed-batch	24.9	4.6	18.8	1200	Wong et al. (2009)
	BPED fed-batch	34.2	5.5	9.7	370	This work

BiotechnolLett (2010Lett ( ) 32:1053Lett ( -1057

Acknowledgements The authors would like to thank the UK Engineering and Physical Sciences Research Council (EPSRC) for support of the multidisciplinary Biocatalysis Integrated with Chemistry and Engineering (BiCE) programme (GR/ S62505/01). Financial support from the 13 industrial partners supporting the BiCE programme is also acknowledged. MW would also like to thank the EPSRC and EKB Technology Ltd for financial support (EngD studentship from the UCL Industrial Doctorate Training Centre (IDTC) in Bioprocess Leadership).