Vincent Rious

Jean-Michel Glachant

Yannick Perez

Philippe Dessante

The role of transmission investment in the coordination between generation and transmission in the liberalized power systems

This paper examines how transmission coordinates with generation to the long term in a liberalized power system. We rely on a modular analysis to separate the mechanisms of coordination between generation and transmission of electricity into distinct modules. The governance structure of transmission completes this analysis framework. We then show that in a logic of complementarity, this governance structure influences the options that TSO implements to manage effectively power flows. Although locational signals are necessary to guide the installation of new power plants, the governance structure explains that investment in network may be the only effective method of longterm coordination between generation and transmission.

Introduction

In liberalized power systems, to ensure non-discriminatory access to transmission network, it was needed to unbundle vertically competitive generation and the natural monopoly of transmission network. Unbundling activities previously integrated in a vertical and horizontal monopoly creates problems of coordination between generation and transmission investments. We show that transmission investment is the mechanism that will effectively realize this new coordination to the long term in a liberalised power system.

Facing this situation, [START_REF] Lévêque | Transport Pricing of Electricity Network[END_REF] has shown that a centralized authority should send locational signals to generators to replace the traditional coordination. This centralized authority is called the Transmission System Operator (TSO). Therefore, we expect that the TSOs implement the best existing signals that should lead to efficient coordination between generation and transmission investments. But the study of TSOs shows that those who implement the more incentive locational signals are not those who experience the best coordination with generation. How to explain this paradox?

To answer this question, in the first section, we propose and build an ideal TSO. We will use this ideal TSO as a benchmark for the study of coordination between generation and transmission of real TSOs. In the second section, we show that the incentive structure of the ideal TSO is difficult to achieve because the governance of electricity transmission assets influences the implementation of power flow management. Finally, in the third section, we apply this modular and organisational framework to compare two TSOs, 1° Pennsylvania, Maryland and New Jersey (PJM), and 2° National Grid. Then we will see what the central mechanism to coordinate generation and transmission of electricity is in real organizations.

1-Ideal organization of a TSO

To manage power flows, the Transmission System Operators realize three main tasks that range from very short term (a few minutes to several hours) to the very long term (5 to 20 years) [START_REF] Brunekreeft | Electricity transmission: an overview of the current debate[END_REF]. These three missions are: i) the management of short run power flow externality on the transmission network; ii) the development of transport capacity, and iii) coordination with neighboring interconnected systems. To study the multiplicity of real grid operators, we have shown in [START_REF] Rious | The diversity of design of TSOs[END_REF] the relevance of a modular analysis framework à la Clark and [START_REF] Baldwin | Design Rules -The Power of Modularity[END_REF] and [START_REF] Wilson | Architecture of the power markets[END_REF] to classify the existing empirical diversity of TSOs. Here we need only to focus on two missions: the short run management of power flow externality and the development of transmission network. If we choose the optimal option for each of these two missions, an ideal TSO can be defined. It is a combination of "nodal pricing" and, under a benevolent regulation, of the minimization of the total cost of network (that is to say, to minimize the sum of the congestion cost and the investment cost of the network). We will detail these two points and see why nodal pricing and long-term development of the transmission network are the building blocks of the optimal organization of an ideal TSO.

First, the optimal short run management of power flow externalities is obtained with the system of nodal pricing of energy. [START_REF] Schweppe | Spot Pricing of Electricity[END_REF] show that an efficient power dispatch can be achieved through a system of nodal pricing of electricity whose clearing is constrained by the externalities associated with congestion of powerlines. This method has incentive virtues superior to those of the two other well-known power flow management schemes, zonal pricing4 and redispatch5 . Thus nodal pricing determines a price of energy for each node of the network. These prices indicate the nodes where it is preferable to produce or consume one more megawatt taking into account the constraints of network capacity.

Figure 1 illustrates nodal pricing on a simple two-node congested network. There is only generation connected to the first node S and its cost is low. To the second node D, there is a quantity Q of inelastic load and also generation whose cost is high. These two nodes are linked by a single powerline SD whose transmission capacity is K. If we ignore the limited capacity of powerline when clearing the market, generation to node S is sufficient to supply load (to node D). The equilibrium price is unique and is P E . This equilibrium induces a flow on line SD greater than the available transmission capacity K, that is to say K < Q. As a consequence, this equilibrium is not technically feasible. To incentivize generation to the nodes S and D to take into account congestion on the line SD, with nodal pricing, the generator at node S is paid a price P S below the equilibrium price without congestion P E and the generator at node D is paid P D a price higher than P E . In this case, the difference in value between the nodal prices reflects the social value of network externalities. This difference creates a surplus for the TSO called "congestion rent". And the constraints of network capacity limit the maximization of social welfare by a quantity called "congestion cost". The congestion rent is represented on Figure 1 by the grey rectangle and the congestion cost by the dark grey trapezoid.

Figure 1 Graphical representation of nodal pricing on a congested two-node network

The second mechanism of coordination between generation and transmission is the long-term development of the power transmission grid. In theory, the management of power flow externalities can inform the TSO and the users of the network on the constraints related to the current state of operation of the network. But to the long run the TSO should also make efficient network investments to eliminate all the constraints on the grid that are economically excessive. If one considers by simplification that the TSO is benevolent and efficient, it must invest in order to reduce the social costs caused by the externalities of use of the network and so, in an equivalent way, to maximize social welfare.

Considering this definition of the ideal TSO, the observation of the modes of organizations of real TSOs reflects a large variety of combination of suboptimal management schemes of power flows. This variety can be explained beyond the technical constraints of power flow management by the different characteristics of the governance structures of the transmission grid.

2-Complementarities between the governance structure and the management of electricity flows

In the liberalized power system, the TSO can take different organisational forms or governance structures, according to whether he owns the property rights of the network he manages, and according to the form of regulation that is applied to this monopoly. The governance structure of the network modifies the incentives that the TSO can perceive from the management of power flows. In this section, first of all, we define the governance structure of the network by presenting its main components. Then, we show that the governance structure of the network influences the options that the TSO implements to manage power flows.

2-a) The governance and the incentive regulation of the network

The way the transmission grid is unbundled constitutes the ground of the governance structure of the network. Because it determines the degree of incentives that can be introduced into the regulation of this monopoly. Unbundling the transmission grid from competitive activities like generation or supply is currently considered essential. This vertical unbundling is of two degrees of intensity. The first level always includes the unbundling of short run system operation. Indeed, the withdrawal of system operation from incumbents is rather easy to impose in a process of liberalization because this activity represents a relatively small volume of investment and employment. To the contrary, the second degree of unbundling for ownership of the power grid depends on the possibility of forcing the incumbents to cede their network assets. This level of vertical unbundling is more difficult to produce at the time of competitive reforms. Indeed a pure generator faces competitive pressures and uncertainty from electricity markets. While the network is a source of regulated revenue that is guaranteed and recurring. This secured source of revenue is then very interesting and attractive for a generator in electricity markets where the companies are also judged on their financial performances. 6The choices of the level of network unbundling can also be influenced by the interactions so called "border effects" between interconnected power systems. If ownership and operation of a grid on a continental scale are strongly fragmented between a lot of distinct TSOs, the loop flows between these TSOs create many border effects that are difficult to deal with and that can reach critical values for the reliability of the system. Then, operation and ownership unbundling of the network in each the TSO's zone may not be sufficient to internalize the "border effects" and solve the associated problems. A remedy is then to remove the "System Operation" part from incumbents and to recompose this function on a wider geographical area including several electric zones, under the operational authority of a new Independent System Operator (ISO). The horizontal integration of system operation on wide zones then allows to internalize the border effects between the previous zones of the incumbents [START_REF] Costello | Interregional coordination versus RTO mergers: a cost-benefit perspective[END_REF]PJM, 2004b).

One can now distinguish two main families of TSOs in terms of degree of unbundling: the "heavy" TSO and the "light" TSO. A "heavy TSO" owns the network infrastructures that he operates. A "light TSO" does not own the network infrastructures that he operates. These modalities of grid unbundling are thus important at the same time to understand the efficiency of the governance of the transmission network but also its regulation as a monopoly. In the case of a "heavy" TSO, the regulator can impose an incentive regulation on the controllable costs of the network to set the income of this monopoly. Indeed, the potential financial risks of an incentive regulation are acceptable for a "heavy GRT" in terms of assets, equity and revenues. To the contrary, it is difficult for a regulator to incentivize strongly a "light TSO" because of its weak financial standing (few assets, little equity, low revenue). This is why the "light TSOs" are usually non-for-profit organizations, partly self-regulated by the market participants in its zone (Barker and al., 1997). 7 .

2-b) The governance of the network and the management of power flows are complementary

A TSO does not have the same incentives to manage and develop its network depending on the combination between its governance structure and its method to manage short-term externalities. Depending on the implemented method, the management of externality can generate rent or cost for the TSO. Nodal pricing effectively allows an efficient dispatching of generation taking into account the network constraints. But congestion rent arising from this method gives a counter-incentive signal to the TSO for its own investment decisions. Indeed, nodal pricing can incentivize a TSO maximizing its profit to make congestion last (Pérez-Arriaga and Al, 1995). A TSO that internalizes power flow externalities with nodal pricing should then be subject to a more demanding regulation to ensure that the maximization of the TSO's profit is in line with the maximization of social welfare.

On the contrary, the method called "redispatch" is considered inefficient to internalize the power flow externalities because the TSO then deals with congestion out of the day-ahead electricity market. In this case, no short-run locational signal is transmitted to the users of the network who cannot then make efficient use of the transmission capacity. But this method has the advantage that the TSO directly bears the congestion cost arising from system operation. Consequently, the TSO can here maximize its profit by comparing the social cost of short term congestions with the long-term cost of investment and maintenance of the grid and thus naturally maximizes social welfare.

The structure of governance of the network and the design of the modules of power flow management must then be seen like a global system. The theoretical perfection would be to combine the options of the ideal TSO with a perimeter of heavy TSO, unbundled from generation and regulated with a strong but also incentivizing regulator. However the complementarities between the governance structure and the modules of power flow management lead to sub-optimal choices in the design of these modules. Compromises must then be realized between the control of the costs of the network and the design of the modules of power flow management.

The study of two TSOs will show in the following section that their structure of governance influences the choice of the mode of coordination between generation and transmission. Thus some TSOs favor internalization of externality by scarcity pricing with given network capacity, while others act directly on the cause of externality by investing and increasing the transmission capacity.

3-The comparison of two TSOs of reference: PJM and National Grid

The objective of this part is twofold. First, we study the consequences of the complementarities on TSOs of reference to explain their choices to manage flows of electricity. We will proceed in two stages. First of all, we will compare the options of power flow management implemented by each TSO with those of the ideal TSO. Then, we will show how the structure of governance of the network constrains the implementation of these options. We show finally that the structure of governance can correct the failure of some suboptimal options of coordination.

From this comparison, the second objective of this section is to show that the effects of complementarities brought by the governance of the network alternatively focus on the choice between "internalize externality of use of the network related to congestion" or "to increase the capacity of the network to treat the cause of congestion". Then we will show that it seems very difficult to carry out these two tasks jointly.

3-a) Comparison of real TSOs to the ideal TSO

Now we will use our construction of ideal TSO as a point of comparison for the study of real TSOs. We consider two TSOs of reference in the international experiments of liberalization of the power systems.

The first, PJM, is a light TSO structured as a non-for-profit organization that operates in the North-East of the United States. PJM is recognized as a model because the nodal pricing enabled him to extend its area of responsibility over a wide part for the North-East of the United States and to become thus the TSO that manages the higher peak load in the world [START_REF] Joskow | Patterns of transmission investment[END_REF]. In spite of the implementation of the best method of internalization, the congestion cost of this zone strongly increased until 2006. In fact, before April 2004, PJM didn't take into account the possibility to reduce congestion cost in the planning of network investments on its zone. The network investments were made only for technical reasons of reliability.

The second model is the TSO National Grid. It is a heavy TSO, owner of the network he operates in England and Wales. He is unbundled from generation. He operates, maintains and develops the network of England and Wales. National Grid is a private company, quoted on the stock exchange, but regulated by the British regulator of energy, the OFGEM8 [START_REF] Joskow | Patterns of transmission investment[END_REF]. This TSO is often quoted as an example for its efficiency in the management of the network within the framework of a liberalized power system [START_REF] Rossignoli | Transmission: The Critical Link Delivering the Promise of Industry Restructuring to Customers[END_REF]). While at the same time National Grid operates its system with redispatch and this option of congestion management does not internalize the externalities of use of the network. But the practical modalities of regulation applied to this monopolist for his system operation and the development of its network push it to invest in order to minimize congestion cost. Its congestion and investment costs have then be considerably reduced.

We note that these two TSOs do not apply the combination of coordination mechanisms of the ideal TSO. And this, although they are both recognized at the international scales for the management of their network. In addition, the trend of congestion cost of TSOs is not coherent with the locational signals used by each one of these TSOs. Indeed, it is paradoxical that PJM sees congestion increased whereas its locational signals are very incentive. In the same way, it is paradoxical that National Grid has a good control of congestion, whereas its locational signals are theoretically weaker.

In the following table, we summarize the comparison of these two TSOs, PJM and National Grid, with the ideal TSO, as well as the evolution of their congestion costs.

Table of comparison of different

3-b) Governance of the network and consequences on the options of congestion management

The structure of governance of the network that completes our initial framework of analysis makes it possible to explain the options implemented by the TSOs and their performances to coordinate generation and transmission of electricity. In particular, the type of network unbundling is central because it determines the most suitable regulation that can be applied to the TSO.

In the case of PJM, the grid remains the property of incumbents. The structure of "light TSO" of PJM has the advantage that it can be easily extended to integrate new zones in its market. This extension is all the more efficient as it is carried out thanks to nodal pricing of energy. The use of this method proves to be obligatory in the case of a light TSO like PJM because, by nature, PJM is insensitive to the congestion cost or the congestion rent resulting from the short run signals. Nevertheless, nodal pricing did not prevent a recrudescence of congestion on the zone of PJM.

The increase of congestion revealed in fact an insufficient coordination between generation and transmission within PJM. This problem ended up by drawing attention of the federal regulator of energy, the FERC (PJM, 2004b;[START_REF] Joskow | Transmission Policy in the United States[END_REF]. Thus, it is only under the pressure of the FERC that PJM defined the concept of "Economic Planned Transmission Facilities" [START_REF] Joskow | Patterns of transmission investment[END_REF]. Without the constraint of the regulator, PJM would probably not have taken this initiative because its statute of light TSO does not incentivize him to do it. Indeed, the heart of its activity is the short-term management of grid and it is thus the extension of this activity of system operation which guides the development of PJM.

Conversely, when the TSO owns the network like National Grid, the development of the infrastructure is its core activity. Network ownership then makes it possible to impose him an incentive regulation. Then, making him bear even only in part the congestion cost arising from redispatch incentivizes him to develop the network efficiently. Indeed, thanks to the structure of governance of heavy TSO of National Grid, an incentive regulation allows to control the congestion cost and, to a lesser extent, the investment costs of the network. This incentive regulation allows to compensate for the theoretical failures of redispatch related to the absence of internalization of externality. The regulation of system operation and of network ownership proposes to National Grid an arbitrage between the costs of system operation in the short and medium term and the investment costs of the network [START_REF] Joskow | Patterns of transmission investment[END_REF]. In this framework, the incentive regulation of system operation prompts National Grid to arbitrate between the congestion cost and transmission investments of small sizes (with short periods of return on investment). While the long run budget constraint incentivizes National Grid to arbitrate between the investments of small size and the investments of bigger size, the latter being able to be less expensive thanks to the economies of scale that characterize power grid.

3-c) Governance of the network and coordination between generation and transmission

Our analysis makes it possible to show that, to deal with congestion, light TSOs and heavy TSOs choose basically different methods. The light TSO like PJM focuses on the internalization of congestion in a price system, while the heavy TSO as National Grid concentrates on the development of the network.

The structure of governance of the network influences coordination between generation and transmission. Depending on its form, the governance incentivizes the TSO to focus either on internalization of congestion (with given transmission capacity) or on the development of the network (in order to treat the cause of these congestions). In practice, the governance structure of the network makes it difficult to reconcile efficiently and simultaneously these two approaches of treatment of externalities.

Considering the performances of PJM and National Grid, we note that the module of network investment holds a central place in coordination between generation and transmission. Even if the system of PJM is best equipped in locational signals, the evolution of congestion cost on its zone shows that the investments in network were insufficient. Then coordination between the investments in generation and network was not satisfactory. Conversely, National Grid is more concerned with the long run coordination with generation. That can be easily understood since he owns the network and prefers to develop it to eliminate congestion. 9But considering the trend of congestion cost, the strategy of developing the network is more efficient. Of course, the locational signals are necessary to incentivize the location of power plants. But the generators have strong constraints of location to settle their new power stations. The primary energy source must be easily accessible. For the thermal power plants (with gas, coal or nuclear power), a river with an important flow is also necessary. Lastly, the generators must find lands that fit with these criteria at a reasonable price. These constraints for the generators lead to durable congestions for which the only solution is to develop the network.

The network investment then holds a central role in long-term coordination between generation and transmission, and this for two reasons. Firstly, the structure of governance of the network can result in setting up little or no coordination signals. As a consequence, the coordination signals do not guarantee that the cause of congestion, related to the lack of capacity of the network, is treated.

Conclusion

We have shown that the complementarities between the structure of governance of the network and the modules of management of power flows influence coordination between generation and transmission of electricity, and this in two manners. First of all, the structure of governance of the network defines the degree of unbundling of the TSO from the rest of the electric system, which modifies the incentives that he can perceive in the various options of power flow management of flows. Then, depending on the structure of governance, only the sub-optimal schemes might be applied, in particular when the optimal options are counterincentive for the TSO in the configuration of its governance. But the structure of governance can also correct some failures of these sub-optimal methods. The conclusions of our analysis are thus more moderate than those of other studies [START_REF] Boucher | Towards a common European Electricity Market -Path in the right direction… still far from an effective design[END_REF][START_REF] Ehrenmann | Inefficiencies in European congestion management proposals[END_REF]. Because they show that it is still useful to study the effects of these options on coordination between generation and transmission. Then, the study of PJM and NGC in terms of complementarity showed that the module of investment of the network has more influence on coordination between generation and transmission than internalization of externalities.

Internalization of the externalities of use of the network is admittedly needed to effectively coordinate the production and the short-term and long-term transmission electricity. But, since the long term location of generators generates durable congestions on the network, only the development of transmission capacity brings a satisfactory coordination of these two activities. The module of investment of the network is thus the heart of coordination between generation and transmission of electricity. The complementarities show that the investment in network can be the only effective process of coordination between generation and transmission.

  (2006) and[START_REF] Rossignoli | Transmission: The Critical Link Delivering the Promise of Industry Restructuring to Customers[END_REF]

Assistant Professors at Supélec, Department of Power Systems and Energy, 3 rue Joliot-Curie, 91192 Gif-sur-Yvette CEDEX

Director of the « Florence School of Regulation » et of the « Loyola de Palacio » chaire at the European University Institute.

Tenure professor at the University Paris-Sud 11, ADIS-Groupe Réseaux Jean Monnet and associated researcher at the « Loyola de Palacio » chaire at the European University Institute.

Zonal pricing is similar to nodal pricing but only the most important network constraints are internalized in the pricing system of electricity market. The other minor constraints are managed by redispatch. This simplification from thousands of nodes to few zones is very sensitive to errors and gaming by generators.

When congestions are managed by redispatch, they are not internalized in the electricity market. The TSO manages congestion after the market clearing by modulating production of the adequate power stations. Redispatch thus does not emit any locational signal, except for the modulated power stations.

Moreover, ownership of the network can make it possible for the generators to protect strategically their generation portfolios in the development plans of the network.

One will notice that the good functioning of this principle of self-regulation is efficient only under the assumption that there is no risk of collusion or capture of the TSO by a single group of interest[START_REF] Boyce | Governance of electricity transmission systems[END_REF].

Office of Gas and Electricity Markets

This is also true when the locational signals are ineffective.


domain: sdv chim

text:
Matthew P Mccusker

Daniela Alves Ferreira

Donal Cooney

Bruno Martins Alves

Séamus Fanning

Jean-Marie Pagès

Marta Martins
email: mmartins@tcd.ie

Anne Davin-Regli

Seámus Fanning

Umr_Md1

Modulation of antimicrobial resistance in clinical isolates of Enterobacter aerogenes: A strategy combining antibiotics and chemosensitisers

Keywords: Chemosensitisers, efflux pump, fluoroquinolones, multidrug resistance, permeability, combination therapy

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Acute hospital-acquired urinary tract infections are mainly associated with Escherichia coli [START_REF] Wang | Urinary tract infections[END_REF]. However, pathogens such as Enterobacter, Klebsiella, some Proteus species and enterococci can also be involved in cases of cystitis and pyelonephritis [START_REF] Brooks | Quantifying the Evolutionary Conservation of genes encoding multidrug efflux pumps in the ESKAPE Pathogens to identify antimicrobial drug targets[END_REF]. Enterobacter represents with Klebsiella the prominent enterobacterial members of the ESKAPE group that encompasses the main MDRs described in the most worrying/complicated infections in hospitals [START_REF] Masi | Antimicrobial resistance and drug efflux pumps in Enterobacter and Klebsiella. In: Efflux-Mediated Drug Resistance in Bacteria: Mechanisms, Regulation and Clinical Implications[END_REF].

In 1999, Enterobacter aerogenes represented 48.7% of the extended-spectrum betalactamases strains, due to the dissemination of TEM-24 [START_REF] Bosi | Most Enterobacter aerogenes strains in France belong to a prevalent clone[END_REF]. In 2007, the numbers decreased, being replaced by E. coli CTX-M producers [START_REF] Anastay | Epidémiologie des bêtalactamases à spectre étendu (BLSE) chez les entérobactéries dans un hôpital du sud de la France, 1999-2007[END_REF][START_REF] Vonberg | The endemic situation of Enterobacter aerogenes and Enterobacter cloacae: you will only discover what you are looking for[END_REF]. It is known that during therapy, strains acquire resistance to β-lactams [START_REF] Bosi | Most Enterobacter aerogenes strains in France belong to a prevalent clone[END_REF][START_REF] Anastay | Epidémiologie des bêtalactamases à spectre étendu (BLSE) chez les entérobactéries dans un hôpital du sud de la France, 1999-2007[END_REF][START_REF] Vonberg | The endemic situation of Enterobacter aerogenes and Enterobacter cloacae: you will only discover what you are looking for[END_REF][START_REF] Giamarellou | Multidrug resistance in Gram-negative bacteria that produce extendedspectrum beta-lactamases (ESBLs)[END_REF]. At present, E. aerogenes is the fourth most frequently isolated bacteria in the clinical setting. This prevalence has increased greatly due to the introduction of extended-spectrum cephalosporins and carbapenems [START_REF] Giamarellou | Multidrug resistance in Gram-negative bacteria that produce extendedspectrum beta-lactamases (ESBLs)[END_REF][START_REF] Arpin | Extended-spectrum beta-lactamase-producing Enterobacteriaceae in community and private health care centers[END_REF][START_REF] Thiolas | Successive emergence of Enterobacter aerogenes strains resistant to imipenem and colistin in a patient[END_REF]. This has contributed to the emergence of pandrug E. aerogenes isolates resistant to last-line antibiotics.

Moreover, clinical strains exhibiting efflux activity are resistant to β-lactams, quinolones, tetracyclines and chloramphenicol [START_REF] Pagès | Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment[END_REF][START_REF] Malléa | Alkylaminoquinolines inhibit the bacterial antibiotic efflux pump in multidrug-resistant clinical isolates[END_REF][START_REF] Masi | Production of the cryptic EefABC efflux pump in Enterobacter aerogenes chloramphenicol-resistant mutants[END_REF][START_REF] Chevalier | Inhibitors of antibiotic efflux in resistant Enterobacter aerogenes and Klebsiella pneumoniae strains[END_REF]. Approximately 40% of MDR clinical strains have an active efflux [START_REF] Chevalier | Identification and evolution of drug efflux pump in clinical Enterobacter aerogenes strains isolated in 1995 and[END_REF].

Clinical isolates of E. aerogenes that overexpress efflux pumps in combination with loss of porins have been reported in numerous studies [START_REF] Pagès | Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment[END_REF][START_REF] Lavigne | Membrane permeability, a pivotal function involved in antibiotic resistance and virulence in Enterobacter aerogenes clinical isolates[END_REF]. The study conducted by Doumith et al. also showed that carbapenem resistance in Klebsiella and Enterobacter was exclusively due to combinations of ß-lactamases with impermeability caused by loss of outer membrane proteins, where efflux was not implicated [START_REF] Doumith | Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK[END_REF]. Taken these studies into account, reduction of membrane permeability through the loss of porins together with increased expression of efflux pumps can constitute a survival response involved in bacterial adaptation under stress [START_REF] Masi | Structure, Function and Regulation of Outer Membrane Proteins Involved in Drug Transport in Enterobactericeae: the OmpF/C -TolC Case[END_REF][START_REF] Bolla | Membrane permeability and regulation of drug 'influx and efflux' in enterobacterial pathogens[END_REF]. This is frequently linked with alteration of antibiotics or modification of the drug targets in multidrug resistant (MDR) isolates [START_REF] Moreillon | Bacterial resistance to antibiotics[END_REF]. In E. coli, E. aerogenes, K. pneumoniae and Salmonella enterica several genes and external factors are involved in the emergence of multidrug resistance [START_REF] Bolla | Membrane permeability and regulation of drug 'influx and efflux' in enterobacterial pathogens[END_REF][START_REF] Moreillon | Bacterial resistance to antibiotics[END_REF][START_REF] Paterson | Resistance in gram-negative bacteria: Enterobacteriaceae[END_REF]. The combined action of these mechanisms during an infection confers a decrease in bacterial susceptibility ensuring dissemination and colonisation of the patient and favoring the acquisition of additional mechanisms of resistance [START_REF] Lavigne | Membrane permeability, a pivotal function involved in antibiotic resistance and virulence in Enterobacter aerogenes clinical isolates[END_REF][START_REF] Doumith | Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK[END_REF][START_REF] Masi | Structure, Function and Regulation of Outer Membrane Proteins Involved in Drug Transport in Enterobactericeae: the OmpF/C -TolC Case[END_REF][START_REF] Bolla | Membrane permeability and regulation of drug 'influx and efflux' in enterobacterial pathogens[END_REF].

New antibiotics and novel approaches to treat MDR bacteria have been pursued [START_REF] Kmeid | Combination therapy for Gramnegative bacteria: what is the evidence?[END_REF]. Among these, the development of chemosensitisers to return the bacteria to its susceptibility status is to consider [START_REF] Bohnert | Efflux inhibition by selective serotonin reuptake inhibitors in Escherichia coli[END_REF]. These compounds are usually of synthetic or natural origin, however a new "trend" to modify existing compounds has gained strength in the last years [START_REF] Bohnert | Efflux inhibition by selective serotonin reuptake inhibitors in Escherichia coli[END_REF][START_REF] Mahamoud | Quinoline derivatives as promising inhibitors of antibiotic efflux pump in multidrug resistant Enterobacter aerogenes isolates[END_REF][START_REF] Shiu | A new plant-derived antibacterial is an inhibitor of efflux pumps in Staphylococcus aureus[END_REF][START_REF] Opperman | Characterization of a novel pyranopyridine inhibitor of the AcrAB efflux pump of Escherichia coli[END_REF].

The present study is focused on the modulation of efflux-mediated resistance to fluoroquinolones or chloramphenicol, in clinical isolates of E. aerogenes. The strategy used combined antibiotics and chemosensitisers to assess possible reversal of resistance to the selected antibiotics. Efflux activity was assessed by a real-time fluorometric assay in the presence of these compounds to evaluate their action on the efflux mechanism of resistance.

Materials and Methods

Media, antibiotics, chemosensitisers and dyes

Mueller

Bacterial strains

A random analysis using nineteen E. aerogenes isolates was performed in this study. These bacterial isolates were obtained from several hospital origins from the south of France in order to avoid bias. Nineteen E. aerogenes clinical isolates were identified as follows: EACRO, EA9AB32, EA17AB48, EA29AB151, EA26UR132, EA22SP84, EA132263, EA149399, EA171450, EAPOL13200, EAART15737, EA178LAV, EABAL959, EA23214, EA108418, EA118259, EA122554, EA131538, EA137454 (Table 1). The antibiotic susceptible E. aerogenes ATCC13048 was used as reference strain.

Antibiotic Susceptibility Testing

Antibiotic susceptibility testing was initially determined using the VITEK-2 platform and sensititre plates TM GNX-2F (TREK Diagnostic Systems, LTD; East Grinstead, West Sussex, UK) as per the manufacturers recommendation. Disc susceptibility testing was also performed and resistance reported according to the European Committee on Antimicrobial Susceptibility Testing (http://www.eucast.org/antimicrobial_susceptibility_testing/breakpoints/) validated for E. coli were followed, only for comparison terms [26]. For POL, the breakpoints used were the ones established by the Societe Francaise de Microbiologie, 2007. For COL the results were analysed in comparison with the reference strain and other available literature [START_REF] Landman | Polymyxins revisited[END_REF][START_REF] Lo-Ten-Foe | Comparative Evaluation of the VITEK 2, Disk Diffusion, Etest, Broth Microdilution, and Agar Dilution Susceptibility Testing Methods for Colistin in Clinical Isolates, Including Heteroresistant Enterobacter cloacae and Acinetobacter baumannii Strains[END_REF]. Isolates resistant to three or more different classes of antibiotics were considered MDR. Isolates demonstrating mono or multiple resistances to CIP and CHL were selected for further studies.

Determination of Minimum Inhibitory Concentrations (MIC)

The MIC of antibiotics and chemosensitisers were determined by broth microdilution method [26]. All experiments were carried out in triplicate and in three separate occasions.

Assessment of respiration in the presence of chemosensitisers

To evaluate the effect of the chemosensitisers on the respiration of the isolates, assays were prepared in 96-well plates as for MIC determination with media containing a tetrazolium redox dye. Plates were placed in an OmniLog incubator-reader at 37°C for 24 hours. The reduction of the dye due to respiration was monitored and the results recorded as Omnilog units/time.

PCR amplification and sequencing of quinolone resistance-determining region of target genes

Primers were designed using E. aerogenes ATCC13048 (KCTC2190) (GenBank: CP002824.1) as reference. For isolate 178LAV primer Ec_parE_Rev was used to amplify the QRDR of parE (Table 2). Genomic DNA was extracted using a DNeasy Blood & Tissue kit (QIAGEN). PCR assays were performed as described [START_REF] Dastidar | Role of Phenothiazines and Structurally Similar Compounds of Plant Origin in the Fight against Infections by Drug Resistant Bacteria[END_REF]. PCR products were digested with 1 U DpnI (New England Biolabs) at 37°C for 1 hour before being purified using the Wizard ® SV Gel and PCR Clean-Up System (Promega) and sequenced (Source BioScience LifeSciences).

PCR for cat and cml genes

To verify if the resistance to CHL was due to the presence of cat and/or cmlA genes, a PCR assay was performed [START_REF] Karczmarczyk | Characterization of multidrug-resistant Escherichia coli isolates from animals presenting at a University Veterinary Hospital[END_REF]. The mixture was initially denaturated (4 min at 94°C), followed by 35 cycles of amplification (30 seconds of denaturation at 94°C, 30 seconds of annealing at 55°C for cat and 59°C for cml, 1 minute of elongation at 72°C) and a final elongation step (5 min at 72°C). Amplifications were carried out on a G-Storm GS1 Thermal Cycler, UK.

Reversal of antibiotic resistance

Antibiotics tested were CIP and CHL, which are concerned by efflux-mediated resistance . Briefly, 100 µl of MH broth was distributed into each well of the 96-wells microplate, except for column 12, that contained 200 µl of the highest concentration of antimicrobials.

Serial twofold dilutions were performed. The chemosensitisers, TZ, CPZ and NMP were added at 0.5x MIC and PAßN at 40 mg/L. 5 µl of adjusted bacterial inoculum were inoculated into the corresponding wells. Plates were incubated at 37°C for 18 hours. All assays were conducted in triplicate in separate occasions.

Assessment of permeability/efflux activity

A real-time assessment of the efflux activity was conducted using a modified 96-well microplate method and fluorescence readings of hoechst dye monitored with a FluoroSkan Ascent Fl (Thermo Scientific, Hampshire) [START_REF] Coldham | A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli[END_REF]. Efflux assays were performed by adding 20 µl PAßN or the other chemosensitisers (at three different concentrations) to the cultures monitored previously. Assays were repeated 4 times per isolate.

Modulation of the efflux dynamics -Determination of kinetic parameters

To analyse the kinetics of accumulation promoted by the chemosensitisers (1/5 MIC), the rate of accumulation of Hoechst dye and the time necessary to reach a plateau (designated Pl) were determined. A regression model was applied to the accumulation of Hoechst-dye curve using a logarithmic or semi-logaritmic regression (case dependent). The regression equation was used to calculate the rate of fluorescence.

Statistical analysis

All data were analysed using Microsoft Excel and SPSS Statistics version 20.0. A dendogram representing the antimicrobial susceptibility data was generated using software from Applied Maths (Bionumerics, Version 7.1, Applied Math, Austin, TX).

Results and Discussion

Antibiotic susceptibility testing

The majority of the isolates were resistant to ceftazidime and ticarcillin-clavulanic acid (n=13), followed by resistance to CTX (n=12) (Figure 1). Twelve isolates were also resistant to azetronam. Resistance to CIP (n=11) and levofloxacin (n=10) was also observed.

Resistance to tobramycin and trimetoprim/sulfamethoxazole was recorded in 10 of the isolates. Lastly, nine of the isolates were resistant to CHL. EAPOL13200 and EA131538 exhibited a quasi-pan resistant phenotype with susceptibility to POL.

MIC for selected chemosensitisers

The chemosensitisers, TZ and CPZ were the ones that showed higher inhibitory activity (MIC = 50 to >200mg/L) (Table 3). PAßN and NMP showed lower inhibitory activity, with MIC values between 250 and 2,000 mg/L and 500 and 1,000 mg/L, respectively. The chemosensitisers, OUB, VER, OME and ESO exhibited lower antibacterial activity (MIC > 2,000 mg/L).

Assessment of respiration in the presence of chemosensitisers

Growth curves of all isolates were performed in the presence of varying concentrations of TZ, CPZ, PAßN and NMP to ensure the chemosensitisers did not exert any effect on bacterial respiration. In the presence of 200 and 400 mg/L of the chemosensitisers, there was a clear impact on the respiration of E. aerogenes ATCC13048, over the course of a quasi-growth curve (Figure 2). Similar effect was obtained with the other compounds. In the presence of 100 mg/L TZ and 50 mg/L CPZ there was an extension on the lag phase up to 6-7 hours (Figure 2). At the highest concentrations of PAßN (400, 200 and 100 mg/L), there was a notable reduction in respiration, with similar effects being observed in the presence of varying concentrations of NMP (Figure 2). Importantly, concentrations of these same chemosensitisers used for the reversal assays, did not exert any influence on the respiration of E. aerogenes ATCC13048 (Figure 2).

In the presence of 200 mg/L TZ, isolates EA178LAV, EA122554, EA22SP84, and EA23214 exhibited a delay in the lag phase. After several hours, the respiration was re-established (Figure 3). Similar results were previously reported for Salmonella, suggesting a process of adaptation occurring among specific isolates [START_REF] Spengler | Genetic response of Salmonella enterica serotype Enteritidis to thioridazine rendering the organism resistant to the agent[END_REF].

A clear inhibition of bacterial respiration was obtained for the majority of the isolates at 320 and 160 mg/L PAßN (Figure 4). 80 mg/L PAßN elicit a delay in the lag phase of EACRO, EAART15737, EA178LAV, EA108418, EAPOL13200, EA23214 and EA118259 (Figure 4 A-E, I, J and K). After 5 hours or more of exposure to 40 mg/L PAßN a more pronounced effect on the respiration of EACRO, EA108418, EA122554, EAPOL13200 and EA131538 (Figure 4 A,D,E,H,L) was obtained. The same was obtained with 20 mg/L PAßN for isolates EA137454, EA149399, EAPOL13200 and EA131538 (Figure 4 F,G,H,L).

When NMP was added at the highest concentrations there was a clear inhibition on the respiration of all isolates (Figure 5). At a concentration of 250 mg/L only EAART15737 seemed to be affected (Figure 5B). Isolates EA149399, EAPOL13200, EA23214, EA118259 and EA131538 showed a delay on the lag phase when incubated in the presence of 125 mg/L NMP (Figure 5 G,H,J,K,L).

Selection of antibiotic resistant strains and synergy testing

Based on the resistance profiles and considering the MIC values obtained for CIP and CHL, twelve isolates were selected for further study (Table 1). Isolate EA149399 was only tested for reversal of resistance to CHL due to its susceptibility to the fluoroquinolones tested. CIP and CHL were chosen to assess the possible contribution of efflux to the resistant profile of the isolates [START_REF] Chevalier | Identification and evolution of drug efflux pump in clinical Enterobacter aerogenes strains isolated in 1995 and[END_REF]. All isolates (with the exception of EA149399) had MIC values of CIP between 4 and 64 mg/L. Similarly, for CHL values were between 128 and >512 mg/L when compared with the reference strain (4 mg/L).

Modulation of antibiotic resistance

The effect on resistance level to CIP and CHL was assessed in the presence of CPZ, PAßN and NMP (Table 4).

In the presence of CPZ, CPZ and PAßN, isolate EAPOL13200 had an MIC for CHL of 16 mg/L (>10× reduction in the original MIC >512 mg/L). In the presence of NMP, decrease of resistance to CHL was also obtained with an 8× reduction in the original MIC (from >512 mg/L to 32 mg/L). Isolate EAART15737 showed a reversal of resistance to CHL (MIC from >512 mg/L to 16 mg/L) in the presence of CPZ, PAßN and NMP.

The isolate EA137454 exhibited a reduction of 4× in the MIC of CIP in the presence of CPZ.

In this same isolate, a total reversal of resistance to CHL was observed in the presence of the chemosensitisers. The MIC of CHL was strongly reduced when CPZ, PAßN and NMP were present (reduction > 16×). Since there was a total reversal of resistance to CIP and CHL in the presence of CPZ, it is possible that this isolate has intrinsic mechanisms of resistance that are susceptible to this compound.

For the remaining isolates no significant reductions in the MIC of CHL (≥ 512 mg/L) were observed (Table 4). PCR assays to detect cat [encoding a chloramphenicol acetyltransferase] and cmlA [encoding a phenicol-specific efflux pump] were negative, failing to yield an amplicon from any of the corresponding resistant isolates. Therefore, we can rule out a direct contribution of both CAT and CML enzymes in this resistance. The differences in values obtained for the susceptible restoration levels for EACRO, EA22SP84, EA178LAV versus those obtained for EAPOL13200, EA137454 and others are likely due to other factors. The divergence between the level of susceptibility restored for CHL compared to the level obtained for fluoroquinolones can be associated to ability of chemosensitizers to restore antibiotics susceptibility. This effect depends of the antibiotic chemical structure, their respective affinity to AcrB pockets and the relative affinity of chemosensitizers for AcrB pockets [START_REF] Ramaswamy | Molecular rationale behind the differential substrate specificity of bacterial RND Multi-Drug transporters[END_REF][START_REF] Zwama | Multiple entry pathways within the efflux transporter AcrB contribute to multidrug recognition[END_REF]. These findings highlight the differences between the isolates since not all of them responded in the same way to the combinations tested suggesting difference in the expression level of AcrB pump, expression of other efflux pumps, possible mutations inside AcrB changing the ligand affinity, etc.

Target gene mutations

Comparative analyses were done using E. aerogenes ATCC13048 (KCTC2190) as reference.

Amino acid substitutions were identified in a number of target genes, associated with resistance to fluoroquinolones (Table 1). Eleven isolates contained single or multiple substitutions within the GyrA subunit along with either single or double substitutions in GyrB. The most common amino acid substitution identified in 10 of the 11 resistant isolates, was located within the gyrA-encoding subunit and was denoted as T83I (Table 1). In GyrB, the most common amino acid substitution found was E466D (eight of the isolates). These mutations can explain the failure of susceptibility restoration by chemosensitisers when used in combination.

Assessment of efflux activity

All isolates were assessed for their efflux-associated MDR phenotype and ability to accumulate hoechst dye (Figure 6A). In general, all isolates demonstrated the same fluorescence level as the reference strain. From the four tested chemosensitisers the most efficient compound promoting the accumulation of the dye was TZ, followed by NMP and lastly CPZ. In the presence of glucose, there was a lower fluorescence signal detected, reflecting a reduced accumulation of the dye due to the energy provided to fuel the efflux machinery. Similarly, from the 3 concentrations tested for each chemosensitiser, the highest (TZ 200 mg/L; CPZ 100 mg/L and NMP 250 mg/L) were those that resulted in an increased accumulation of the dye. This response was dose dependent. At these concentrations, addition of glucose had no effect, suggesting that the efflux systems were working at saturation and that respiration was impaired.

PAßN was unable to induce a significant increased accumulation of Hoechst-dye. In the presence of 40 mg/L PAßN, E. aerogenes ATCC13048 and EA149399 were the only isolates that showed a small rate increase in dye accumulation (Figure 6C). This observation is in line with data obtained from the reversal assays wherein in the presence of PAßN, the majority of the isolates did not show any increase in susceptibility to the antimicrobials tested.

At the highest concentration of NMP (250 mg/L) there was a noted interference/possible quenching effect on the fluorescence of Hoechst-dye and this effect was more marked with glucose (Figure 6B).

Modulation of the efflux dynamics -determination of kinetic parameters

CCCP is able to dissipate the proton motive force which is the energy driving force of AcrAB efflux pump when the other like PAßN or NMP can block or saturate the AcrB transport for some antibiotics [START_REF] Nikaido | Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria[END_REF]. An effect of CCCP, PAßN and NMP on the accumulation of the dye was noted with isolates EA149399, EAPOL13200, EAART15737, EA137454 and EA118259 (Table 5). Interestingly, the maximal restoring of CHL susceptibility was obtained with PAßN-NMP on isolates EAPOL13200, EAART15737 and EA137454. This observation suggests that the activity of efflux pump as transporter of CHL and dye is efficiently targeted by the chemosensitisers in these isolates.

Regarding the mechanism and taking into account the effect of glucose, this becomes more complex. Comparing the effect of PAßN alone versus PAßN+glucose (or NMP alone versus NMP+glucose) with the effect of CCCP alone versus CCCP+glucose, it is clear that CCCP is a real efflux energy un-coupler and its uncoupling impact can be partly restore in the presence of glucose. The results obtained with PAßN, NMP and CCCP were of the same order of magnitude supporting some additional effect for CPZ under these conditions. Regarding CPZ, the permeabilizing effect reported for phenothiazines on biological membranes can boost the observed accumulation in association with the chemical properties of Hoechst-dye [START_REF] Dastidar | Role of Phenothiazines and Structurally Similar Compounds of Plant Origin in the Fight against Infections by Drug Resistant Bacteria[END_REF].

Regarding CPZ, an effect is observed with strain EA122554. Taken together, these data illustrates the impact that chemosensitisers can have on the accumulation of dyes, which can be different from the effect on antibiotic activity.

Conclusion

Overall, we explored the potential utility of a series of chemosensitisers in combination with antimicrobials to revert antimicrobial resistance in clinical isolates of E. aerogenes. The isolates studied were also characterized with target gene mutations identified. These genotypes most likely underpin our inability to reverse the resistant phenotypes in the presence of chemosensitisers. For some of the isolates, permeability differences were noted that could contribute to their resistant profile.

Interestingly, the presence of an energy-dependent efflux of Hoescht-dye, CCCP sensitive and activated by glucose, is demonstrated in strains EACRO, EA149399, EAPOL13200, EAART15737, EA118259, EA137454. In strains EAPOL13200 and EA137454, PAßN can impair this efflux when NMP is able to alter its activity in strains EAPOL13200 and EA149399. Regarding these effects, we may hypothesize that PAßN and NMP can have same activity behavior in strain EA149399 and exhibit different action in strains EAPOL13200 and EA137454. These differences in the inhibitor activity may be due to different affinities of Hoescht-dye, PAßN and NMP for the binding sites located inside efflux pump or by the steric hindrances caused by the fixation in the AcrB pockets [START_REF] Vargiu | Multidrug binding properties of the AcrB efflux pump characterized by molecular dynamics simulations[END_REF][START_REF] Ruggerone | RND efflux pumps: structural information translated into function and inhibition mechanisms[END_REF].

The more important point is the correlation between accumulation studies and restoration of antibiotic activity. For fluoroquinolone, it is clear that the presence of target mutations in the various isolates drastically impair the recovery of ciprofloxacin susceptibility whatever the chemosensitizers used. About CHL, the resistance is efficiently reversed by the three chemosensitizers in three strains for which an effect of CCCP on Hoescht accumulation has been evidenced. This indicates that CPZ, PAßN and NMP are able to block the CHL efflux in these strains and restore a partial or complete susceptible phenotype. Interestingly, no restoration of CHL susceptibility is obtained for the strain EA149399 for which an efflux CCCP, PAßN and NMP sensitive is observed with Hoescht. The divergence observed between the susceptibility restoration and the dye accumulation is probably due to the difference of affinity and binding constant for the pump, and/or the presence of the antibiotic target which initiates the bacterial death when the dye assay is only based to DNA or membrane association. This divergence dye-antibiotic must be taken into consideration when assessing the contribution of efflux in clinical isolates and the evaluation of new adjuvants or chemosensitizers. In this way, the use of the recent concept "structure intracellular concentration activity relationship", or SICAR will be more efficient to study the correlation accumulation-activity of clinically-used or new antibiotics and the potential of new adjuvants/chemosensitizers [START_REF] Masi | Mechanisms of envelope permeability and antibiotic influx and efflux in Gram-negative bacteria[END_REF].

Figure legends

Figure 1: A heat-map showing the antimicrobial susceptibility profiles for E. aerogenes ATCC13048 along with 19 clinical isolates (see also Table 1). The antimicrobial compounds clusters are grouped according to their class. Legend. Ciprofloxacin (CIP); Chloramphenicol (CHL); (-) not determined since the isolate is susceptible to the antibiotics. NA -not applicable.

Significant differences in the MIC (considered as > 4-fold) when in the presence of the chemosensitisers are marked (bold). Isolates highlited in bold were selected for the checkerboard assay.

  -Hinton (MH) broth, MH agar (MHA), Phosphate Buffered Saline (PBS) solution, ciprofloxacin (CIP), chloramphenicol (CHL), cefotaxime (CTX), thioridazine (TZ), chlorpromazine (CPZ), ouabain (OUB), verapamil (VER), phenylalanine-Arginine-βnaphthylamide (PAßN), Omeprazole (OME), esomeprazole (ESO), 1-(1-naphthylmethyl)piperazine (NMP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and Hoescht dye solution (stock 20 mM). They were all purchased from Sigma (Ireland).

Figure 2 :

 2 Figure 2: Bacterial respiration trace profiles for E. aerogenes ATCC13048 alone and in

Figure 3 :

 3 Figure 3: Bacterial respiration trace profiles for four clinical E. aerogenes measured in

Figure 4 :

 4 Figure 4: Bacterial respiration trace profiles for clinical E. aerogenes measured in the

Figure 5 :

 5 Figure 5: Bacterial respiration trace profiles for clinical E. aerogenes measured in the

Figure 6 :

 6 Figure 6: Accumulation of Hoechst-dye by: (A) the E. aerogenes isolates tested ; (B) E.

Legend.

  wo/Gluc -without glucose; w/Gluc -with glucose; Thioridazine (TZ); Chlorpromazine (CPZ); Phenylalanine arginine β-naphthylamide (PAßN); 1-(1-naphthylmethyl)-piperazine (NMP); cyanide-m-chlorophenylhydrazone (CCCP). Values are representative of the rate of accumulation of Hoescht dye in the presence of the chemosensitisers and when in the presence or absence of glucose. The grey color scheme indicates a significant effect (ratio without/with glucose >4) of energy (glucose) on the acumulation rate.

Figure 1 .

 1 Figure 1. A heat-map showing the antimicrobial susceptibility profiles for E. aerogenes ATCC13048 along with 19 clinical

Figure 2 .Legend of Figure 2 .Figure 3 .Legend of Figure 3 .Figure 4 .Legend of Figure 4 .Figure 5 .Legend of Figure 5 .Figure 6 :

 223344556 Figure 2. Bacterial respiration trace profiles for E. aerogenes ATCC13048 alone and in

Table 1 . Study collection including E. aerogenes ATCC13048 and 19 clinical isolates and 552 showing their clinical origin; geographical location, year of isolation and mutations in 553 target genes. 554

 1 Legend. MAR -Marseille, AUB -Aubagne, NIMES -Nîmes, nd -not determined. 555

	EA23214	Catheter	NIMES	2005	T83I	S464F	S80I	N477D
	EA178LAV	Urine	MAR	1999	T83I	E466D S80I	N477D
	EA108418	-	MAR	2003	T83I	E466D S80I	N477D
					A67S,	L451F,
	EA137454	-	MAR	2003			S80I	N477D
					T83I	Q465R
	EA122554	-	MAR	2003	T83I	E466D S80I	N477D
	EA131538	-	MAR	2003	T83I	E466D S80I	N477D
	EA118259	-	MAR	2003	T83I	E466D S80I	N477D
	EABAL959	Urine	AUB	2002	nd	nd	nd	nd
	EAPOL13200	Sputum	AUB	2002	T83I	E466D S80I	N477D
					T83Y,
					D87H,
	EAART15737	Rectal swab	AUB	2002	M127L,	R387K S80I	N477D
					S128A,
					N200D
	EACRO	Urine	AUB	2002	T83I	E466D S80I	N477D
	EA9AB32	Sputum	MAR	1994	nd	nd	nd	nd
	EA22SP84	Suppuration	MAR	1995	T83I	E466D S80I	N477D
	EA17AB48	Sputum	MAR	1995	nd	nd	nd	nd
	EA29AB151	Sputum	MAR	1995	nd	nd	nd	nd
	EA26UR132	Urine	MAR	1995	nd	nd	nd	nd
	EA171450	Sputum	MAR	2002	nd	nd	nd	nd
	EA132263	Sputum	MAR	2002	nd	nd	nd	nd
	EA149399	Blood culture	MAR	2002	nd	nd	nd	nd

Table 2 . Target genes, amplification primers and PCR reaction conditions used for characterisation of the isolates.

 2

	Note: Fwd, forward; Rev, revers

Table 4 : MIC values measured in the absence or in thepresence of selected chemosensitisers along with mutations in target genes.

 4

	E. aerogenes		CIP				CHL		FQ resistance genes (mutation)
	strains	-	+ CPZ	+ PAßN	+ NMP	-	+ CPZ	+ PAßN	+ NMP	gyrA	gyrB	parC	parE
	ATCC13048	1	(-)	(-)	(-)	4	(-)	(-)	(-)	(-)	(-)	(-)	(-)
	EACRO	16	8 (2x)	16	8 (2x)	>512	512	512	512	T83I	E466D	S80I	N477D
	EA22SP84	32	32	64 (2x)	16 (2x)	>512	512	512	>512	T83I	E466D	S80I	N477D
	EA149399	1	(-)	(-)	(-)	128	128	128	128	NA	NA	NA	NA
	EAPOL13200	32	16 (2x)	32	16 (2x)	>512	16 (10x)	16 (10x)	32 (8x)	T83I	E466D	S80I	N477D
										T83Y, D87H,
	EAART15737	64	32 (2x)	32 (2x)	32 (2x)	>512	16 (10x)	16 (10x)	16 (10x)	M127L, S128A,	R387K	S80I	N477D
										N200D
	EA178LAV	16	8 (2x)	16	16	>512	512	512	512	T83I	E466D	S80I	N477D
	EA23214	32	16 (2x)	32	16 (2x)	>512	512	512	512	T83I	S464F	S80I	N477D
	EA108418	32	16 (2x)	32	16 (2x)	>512	512	512	512	T83I	E466D	S80I	N477D
	EA118259	32	16 (2x)	32	16 (2x)	>512	256 (>2x)	512	512	T83I	E466D	S80I	N477D
	EA122554	16	8 (2x)	16	8 (2x)	>512	512	512	512	T83I	E466D	S80I	N477D
	EA131538	16	16	32	16	>512	512	512	512	T83I	E466D	S80I	N477D
	EA137454	4	1 (4x)	8	4	>512	1 (>18x)	2 (16x)	2 (16x)	A67S, T83I	L451F, Q465R	S80I	N477D

Table 5 . Accumulation rates measured in a Hoescht-dye uptake assay and shown as a heat-map.

 5

					Chemosensitisers
	Enterobacter
		CPZ		PAßN	NMP	CCCP
	strains
		wo/Gluc w/Gluc wo/Gluc w/Gluc wo/Gluc w/Gluc wo/Gluc	w/Gluc
	ATCC13048	0.7216	0.4996	0.085	0.0832	0.1689	0.153	0.0645	0.1622
	EACRO	0.4966	0.378	0.0411	0.0275	0.0544	0.0204	0.145	0.0058
	EA22SP84	0.5748	0.3964	0.0845	0.0887	0.1047	0.1042	0.0427	0.0833
	EA149399	0.8903	0.643	0.0897	0.0207	0.113	0.0103	0.0969	-0.0009
	EAPOL13200	0.5009	0.3579	0.0065	0.0078	0.0844	0.0206	0.1128	0.0023
	EAART15737	0.6169	0.3714	0.0149	0.0152	-0.2135	-0.1291	0.0667	0.0024
	EA178LAV	0.5871	0.4078	0.0311	0.0248	0.0561	0.0686	0.006	0.0719
	EA23214	0.6285	0.2984	0.0093	0.014	0.018	-0.0026	-0.0035	0.0293
	EA108418	0.7383	0.3999	0.0384	0.0606	0.1035	0.0737	0.0082	0.1094
	EA118259	0.6424	0.3975	0.0804	0.0413	0.1018	0.169	0.0839	0.0134
	EA122554	0.6755	0.1347	0.0834	0.0855	0.1274	0.1007	0.035	0.0832
	EA131538	0.6887	0.4519	0.0326	0.0155	0.1191	0.0766	0.0072	0.0966
	EA137454	0.7409	0.5719	0.1212	0.0172	0.1633	0.1546	0.1753	0.0123

Table

  ). The antimicrobial compounds clusters are grouped according to their class.

	Cephalosporins	Carbapenems	M o n o	FQs	Aminoglicosides Tetracyclines	Miscellaneous
						!

Acknowledgements

A. Davin-Regli, J.M. Pagès, S. Fanning and M. Martins are members of the translocation consortium (www.translocation.eu).

Legend of Figure 1. The dendogram was produced with software from Applied Maths (Bionumerics, Version 7.1, Applied Math, Austin, TX). Resistance profiles of the isolates are color-coded: resistant (red); intermediate (yellow/orange); susceptible (green). Penicillin G was included in the assay and analysis as an internal control (data not shown). E. coli ATCC25922 was included as quality control.

Declarations

Funding: The research leading to the results discussed here was conducted as part of the translocation consortium (www.translocation.eu) and has received support from the


Laurent Pichon
email: laurent.pichon@univ-rennes1.fr

K Yang

Anne-Claire Salaün

A-C Salaün

Experimental validation of the surface state distribution model in the Suzuki theory to qualify the thin film surface materials

Keywords: Suzuki theory, surface state density, field effect conductance, polycrystalline silicon, thin film transistors

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

I. INTRODUCTION

Since the earlier of the 90s, applications based on electronic components manufactured from thin-film semiconductors is widely spread, in particular for visualization systems [START_REF] Someya | TFT active matrix liquid crystal display devices[END_REF][START_REF] Stewart | Polysilicon TFT Technology for Active Matrix OLED Displays[END_REF], and more recently for electronics systems on flexible substrates [START_REF] Chen | Flexible active-matrix electronic ink display[END_REF][START_REF] Inoue | Surface-free technology by laser annealing (SUFTLA) and its application to poly-Si TFT-LCDs on plastic film with integrated drivers[END_REF][START_REF] Kim | Stretchable silicon nanoribbon electronics for skin prosthesis[END_REF]. These semiconductors have poorer properties than those of bulk materials, because of their disordered structure, or because of the appearance of defects induced by their specific uses (mechanical stress…) [START_REF] Lee | Electrical properties correlated with redistributed deep states in a-Si:H thin film transistors on flexible substrates undergoing mechanical bending[END_REF]. The performances of the corresponding applications are strongly related to the thin film transistor (TFT), basic active component constituting the electronic of the system. The development of TFTs based electronics requires relevant diagnostic tools to assess electronic properties of the systems, both strongly related to the active layer/gate insulator interface and active layer qualities of the transistor. In this way, several methods of electrical characterization to qualify TFT manufacturing technologies were developed, in particular for the widely used silicon thin film technology still acting as reference.

Electrical behaviour of thin film silicon layer is different from single bulk silicon due to higher defect density originating from polycrystalline structure. In this case, the electronic transport is strongly affected by the carriers trapping effects of these defects [START_REF] Seto | The electrical properties of polycristalline silicon films[END_REF]. Two main causes of defects are usually invoked: dangling bonds and strained bonds corresponding to deep and shallow level trap states distributed into the band gap respectively. Two types of numerical models for state distributions are then considered: Gaussian distribution with a maximum around midgap and exponential band tailing corresponding to dangling bonds and strained bonds type defects respectively [START_REF] Fortunato | Field-effect analysis for the determination of gap-state density and Fermi level temperature dependence in polycristalline silicon[END_REF][START_REF] Hirae | Energy distribution of trapping states in polycristalline silicon[END_REF][START_REF] Pesic | Degradation of 4H-SiC IGBT threshold characteristics due to SiC/SiO2 interface defects[END_REF]. The corresponding states energy distributions are representative of the crystal quality of the material. In this way, many studies have been devoted to the determination of the density of states (DOS) into the layer and surface state density (N SS ) energy distributions used as diagnostic tools. Methods based on capacitance, resistivity, conductance activation energy and low frequency noise measurements exist to determine these distributions [START_REF] Hirae | Energy distribution of trapping states in polycristalline silicon[END_REF] [START_REF] Yamamoto | Energy distribution of trapping states at grain boundaries in polycristalline silicon[END_REF][START_REF] Dimitriadis | Determination of bulk states and interface states distributions in polycristalline silicon thin-film transistors[END_REF][START_REF] Pichon | Improvement in the determination by 1/f noise measurements of the interface state distribution in polysilicon thin film transistors in relation with the compensation law of Meyer Neldel[END_REF]. However, few methods allow the discrimination of the deep level state energy distribution from the high level one as hyperfine diagnostic tools. Characterization of polycrystalline silicon (poly-Si) and active layer/SiO 2 interface qualities are fundamental for the well understanding of electrical properties of poly-Si thin film based MOS devices.

In this study, active layer/SiO 2 interface quality in poly-Si thin film transistors is characterized by the determination of the densities of defect states into the bandgap using Suzuki's theory model [START_REF] Suzuki | Theoretical interpretations of the gap state density determined from the field effect and capacitance-voltage characteristics of amorphous semiconductors[END_REF]. This theory reports on two models for the determination of the density of states distribution for defects located into the active layer (DOS) and at the interface (N SS ) respectively. In this theory DOS and N SS calculations are based on the surface potential dependence with the gate voltage and on the conductance of the TFT. DOS is classically and commonly determined following Suzuki's theory, and thus refers to the active layer morphology and crystal quality (ie defects responsible of carriers trapping) [START_REF] Lee | Correlation between gap state density and bias stress reliability of nanocrystalline TFTs comparing with hydrogenated amorphous silicon TFTs[END_REF]. However, to our knowledge N SS experimental determination has never been conducted following Suzuki's theory, because in this case the surface potential with gate voltage dependence specifically requires C-V characteristics. Indeed, C-V measurements are difficult to implement for polycrystalline silicon TFTs, especially because of the high defect density into the insulator and at the insulator/active layer interface. To overcome this drawback, temperature measurements of the field effect conductance has been firstly used as alternative approach for N SS determination according Suzuki model. Following this method, our analysis reports on the first experimental validation of the determination of the state distribution at the surface of the poly-Si thin films based on Suzuki's theoretical surface states model, and discrimination of the nature of involved defects is originally shown using this model.

II. EXPERIMENTAL DETAILS

TFTs used in this study are fabricated by a low temperature (≤ 600°C) four masks process, and are fabricated on glass substrate covered with a SiO 2 layer. Active layer is made of nonintentionally doped amorphous silicon (as-Si) deposited by LPCVD (low chemical vapor pressure deposition). Gate insulator is made of silicon dioxide layer deposited by CVD process at atmospheric pressure. Source/Drain regions are built in LPCVD highly in-situ doped as-Si layer using phosphine gas. Source and drain electrodes are made of thermally evaporated aluminium.

Active layer thickness is 150nm, gate insulator is 100nm and 70nm thick for FSPC and LSPC TFTs respectively.

TFTs are issued from two different crystallization techniques of the active layer: Furnace Solid Phase Crystallized (FSPC) poly-Si TFTs and Laser Solid Phase Crystallized (LSPC) poly-Si TFTs.

For FSPC TFTs, a thermal annealing in nitrogen ambient ensures crystallization of the as-Si active layer, and for LSPC TFTs an additional SPC thermal annealing of the active layer is carried out by an Argon laser beam. In addition, for FSPC TFTs channel, Source/Drain are built in one amorphous silicon layer with two differently doped stack regions (the lower part stating as active layer is undoped, and the upper part is highly doped as source /drain regions). For LPC TFTs two amorphous silicon layers are deposited (a undoped one as active layer and a second one highly doped as source/drain regions). More details about the fabrication process of the TFTs are reported in the ref [START_REF] Pichon | Improvement in the determination by 1/f noise measurements of the interface state distribution in polysilicon thin film transistors in relation with the compensation law of Meyer Neldel[END_REF].

The static I-V electrical characteristics of the devices are collected at room temperature using an Agilent B1500A semiconductor parameter analyser. Temperature measurements were carried out from 300 to 400K using a temperature controlled wafer probe system [START_REF] Cordier | Low frequency noise model in N-MOS transistors operating from subthreshold to above regions[END_REF].

Figure 1 shows transfer characteristics plotted in semilog scale for FSPC and LSPC TFTs operating the in linear mode, representative of more than ten of each type of tested devices. The LSPC TFTs exhibits better electrical performances. The on-state current is higher, the threshold voltage (V TH ) is lower and the switching properties (subtrhreshold slope S) are better: V TH = 6.3V and S = 1.4V/dec for FSPC TFTs, V TH = 2.4V and S = 0.4V/dec for LSPC TFTs. In addition, one can remark that no significant gate voltage shift of the transfer characteristics is observed, and that improvement of the electrical properties in LSPC TFTs is mainly related to the subthreshold slope due to improvement of the interface quality.

III. MODEL OF INTERFACE STATE DISTRIBUTION

There are two main complementary approaches based on analytical methods [START_REF] Dimitriadis | Determination of bulk states and interface states distributions in polycristalline silicon thin-film transistors[END_REF][START_REF] Suzuki | Theoretical interpretations of the gap state density determined from the field effect and capacitance-voltage characteristics of amorphous semiconductors[END_REF] or temperature measurements of the current [START_REF] Fortunato | Field-effect analysis for the determination of gap-state density and Fermi level temperature dependence in polycristalline silicon[END_REF] previously established to qualify active layer by determination of the DOS and the N SS in amorphous or polycrystalline silicon TFTs. These methods are based on the analysis of the field-effect conductance, and it has been usually admitted that it is sensitive enough to account for the effect of the silicon film morphology. Following Suzuki's theory [START_REF] Suzuki | Theoretical interpretations of the gap state density determined from the field effect and capacitance-voltage characteristics of amorphous semiconductors[END_REF], the calculations are mainly focused on the distribution of states density into the active layer [START_REF] Lee | Correlation between gap state density and bias stress reliability of nanocrystalline TFTs comparing with hydrogenated amorphous silicon TFTs[END_REF][START_REF] Fortunato | Determination of gap state density in polycristalline silicon by field-effect conductance[END_REF], but the determination of surface state density in relation to surface quality of material has never been experimentally studied. However, the induced surface charge density, Q SS , at localized interface traps is given by [START_REF] Suzuki | Theoretical interpretations of the gap state density determined from the field effect and capacitance-voltage characteristics of amorphous semiconductors[END_REF]:

( ) ( ) 0 exp 1   = -- - -     si s OX SS s s GS FB s si OX G q Q d V V dG d kT d ε ψ ε ψ ψ ψ (1)
where Ψ S refers to the potential at the SiO 2 /Si interface, G 0 is the conductance at the flatband voltage (i.e. Ψ S =0), ε Si the dielectric constant of the silicon, ε OX the dielectric constant of the oxide, d OX the thickness of the gate oxide, d Si the active layer thickness, V FB the flatband voltage, V GS the gate bias, k the Boltzman constant, T the temperature and q the elementary electrical charge.

The induced charge trapped at the interface is given by: ( )

0 0 ( ) F s F E q SS s SS E Q q N E d E ψ ψ + = -∫ (2)
where N SS stands for the energy distribution of surface state density, E F0 = E C(bulk) -E F , with E F the equilibrium fermi level position and E C(Bulk) the conduction band energy. Therefore, the surface state density is deduced from [START_REF] Suzuki | Theoretical interpretations of the gap state density determined from the field effect and capacitance-voltage characteristics of amorphous semiconductors[END_REF]:

( ) ( ) ( ) 0 1 SS s SS F s s Q N E q q q ψ ψ ψ ∂ + ≅ - ∂ (3)
A calculation of Q SS given by ( 1) is firstly needed for the determination of N SS . This approach is based on the use of the experimental variations of the conductance G with the gate voltage V GS , and the knowledge of the dependence of the surface potential Ψ S with V GS . Two approaches have been previously developed. In the first one [START_REF] Suzuki | Theoretical interpretations of the gap state density determined from the field effect and capacitance-voltage characteristics of amorphous semiconductors[END_REF], iterative process with Ψ S (i) = Ψ S (i-1) + dΨ S is used to numerically approximate the relation Ψ S = f(V GS ). In this case the increment of Ψs can be theoretically determined from C-V characteristics, but difficult to plot for polycristalline silicon based MOS capacitor. In the second method [START_REF] Fortunato | Field-effect analysis for the determination of gap-state density and Fermi level temperature dependence in polycristalline silicon[END_REF], the experimental measurements of the drain current with temperature are exploited: the deduced activation energy, E a , makes it possible to state Ψ S dependence on V GS following (see fig. 2):

( ) 0 s F a GS q E E V ψ ≅ - ( 4
)
with E F0 the activation energy for V GS = V FB (corresponding to the minimum of the drain current of the transfer characteristics).

In this study, calculation of N SS has been carried out following

a s GS GS E qd dV V ψ   ∂ ≈ -  ∂   deduced
from the E a = f(V GS ) curves for the FSPC and LSPC TFTs plotted in the figure 3, with dV GS = 50mV. Variation of the conductance is obtained by dG = dV GS (g m /V DS ) for TFTs operating in the linear mode.

According to (1) negative values, due to electrons trapping at defects, were admitted for calculations of Q SS . In this study flatband voltage is determined as the voltage where the drain current is the minimum, and then calculations were carried out with V FB = -4V (E F0 = 0.48 eV) and V FB = -3V (E F0 = 0.52 eV) for the FSPC and LSPC TFTs respectively. Activation energy was determined from the slope of the linear Arrhenius plots Ea = f(1/T).

IV. RESULTS AND DISCUSSION

The plots of the calculated N SS for the two types of TFTs are reported in the figure 4. Values are consistent with previous results obtained by other methods [START_REF] Dimitriadis | Determination of bulk states and interface states distributions in polycristalline silicon thin-film transistors[END_REF][START_REF] Pichon | Improvement in the determination by 1/f noise measurements of the interface state distribution in polysilicon thin film transistors in relation with the compensation law of Meyer Neldel[END_REF]. In addition, the results show that surface states distribution is lower for the LSPC TFTs, more particularly in the lower part of the bandgap. This tendency is also observed for the DOS calculated by the Suzuki's incremental method in the core of the poly-Si active layer (fig. 4. (b)) and confirm that. These results agree with the decrease of the electrical performances (higher threshold voltage and subthreshold slope, lower On-state current) of the FSPC transistor (see fig. 1). This means that the active layer/SiO 2 interface and poly-Si active layer qualities are better thanks to the beneficial effect of the additional laser annealing. Indeed, laser annealing has been commonly used to improve the electrical properties of TFTs due to a decrease of the defect density into the poly-Si active layer [START_REF] Sera | High-Performance TFT's Fabricated by XeCl Excimer Laser Annealing of Hydrogenated Amorphous-Silicon Film[END_REF][START_REF] Khono | High performance poly-Si TFTs fabricated using pulsed laser annealing and remote plasma CVD with low temperature processing[END_REF][START_REF] Angelis | Effect of excimer laser annealing on the structural and electrical properties of polycrystalline silicon thin-film transistors[END_REF]. Moreover, it is noted that the contribution shape of state distribution for the deep states (ie the increase of density when energy level decreases) is more noticeable for LSPC TFT and refers to theoretical Gaussiantype distribution to model defects, because of evident presence of dangling bonds (acting as acceptor like states in our case) at the interface between two materials. These observations agree with a previous work [START_REF] Pichon | Improvement in the determination by 1/f noise measurements of the interface state distribution in polysilicon thin film transistors in relation with the compensation law of Meyer Neldel[END_REF] reporting on the determination by low frequency (1/f) noise measurements of interface state distribution in these polycrystalline silicon TFTs. In this study, noise analysis has been supported by the theory of charge carrier trapping/detrapping due to defects located at the interface.

Both interface and bulk states distributions are mainly related to the spatial distribution of defects either located at the surface and/or in the core of the polycrystalline silicon layer in relation to the crystallization process. Indeed, crystallization of a-Si layer starts at the seed nucleation layer located in the SiO 2 /a-Si layer bottom interface. In this case, grains growth vertically in a columnar type structure, resulting to higher defects density (including grain boundaries) at the surface of polycrystalline silicon active layer (see fig. 5). This grain morphology and structural defects density were previously revealed by scanning electron microscopy study [START_REF] Haji | Mode of growth and microstructure of polycristalline silicon obtained by solidphase crystallization of an amorphous silicon film[END_REF].

Based on the results of a previous numerical study [START_REF] Dimitriadis | Determination of bulk states and interface states distributions in polycristalline silicon thin-film transistors[END_REF], the deep state distribution could be related to defects mainly located at grain boundaries of the poly-Si layer located at the active layer/SiO 2 gate insulator interface. These grain boundaries, usually recognized as places for dangling bonds, act as traps areas and strongly affect the field effect conductance of the transistors [START_REF] Seto | The electrical properties of polycristalline silicon films[END_REF]. Therefore, according to the plots reported in the fig. 4 the SPC laser annealing induces a significant lowering of the strained bonds type defects density close to the interface. However, the reduction is not so significant for LSPC TFT. Two reasons could be invoked to explain it. The first one is because solid phase laser annealing does not present a high degree of crystal quality as usually provided by others laser liquid crystallization process. In this case, crystallization from layer of FSPC TFT do not have the same specifications in terms of morphology and crystal quality because of the crystallization thermal annealing.

Transistors characteristics combined with analysis of energy distribution of defects density based on the Suzuki's surface states model is another way to fully characterize surface of poly-Si layers in relation to its morphology. This method proposed in this study allows discrimination of the nature of surface defects involved in trapping of carriers, and can be extendedly used as hyperfine diagnostic tool in complement of the usual determination of DOS, for surface characterization of non crystalline thin layers.

Figure 1 :

 1 Figure 1: Transfer characteristics of FSPC and LSPC TFTs at V DS = 1V (linear mode).

Figure 2 :Figure 3 :

 23 Figure 2: Energy band diagram of a metal/oxide/semiconductor (MOS) structure with a positive

Figure 4 :Figure 5 : 14 Figure 1 -

 45141 Figure 4: Active layer/silicon dioxide interface states distribution calculated for FSPC and LSPC

liquid to solid phase results to higher crystal quality of the polycrystalline silicon active layer with higher grain size and lower intra-grain defects after re-solidification, leading to better electrical performances of the TFTs [18-20, 22, 23]. The second possible explanation is because at high gate voltages (ie in the strong inversion) conductance of the transistor is rather controlled by access resistance related to the active layer/Drain (Source) region interfaces, and thus responsible of a higher defect density at shallow energy level observed in LSPC TFTs. On other hand, for devices operating from the depleted to inversion mode (ie in the switching region) the conductance is strongly affected by the trapping effects of carriers (at grain boundaries) into the channel region. This means that the state distribution determination is relevant in the lower part of the bandgap. These distributions deduced from the theoretical Suzuki's surface model highlight the separated contributions of the type of defects (see fig. 2). Such distribution behavior related to the deep state defects for the DOS was not observed in previous work [START_REF] Borgne | Electrical properties of self-aligned gate-all-around polycristalline silicon nanowires field-effect transistors[END_REF], and neither for our studied TFT (fig. 4.

(b)). This means that model of calculation of DOS into the bandgap classically used by Suzuki

method is an incomplete view of defects distribution at the surface and into the core of the active layer. Therefore, our results demonstrate that surface states model of Suzuki is a relevant and complementary diagnostic tool for surface characterization of thin film material.

V. CONCLUSION

Capacitance voltage measurements on MOS (or MIS) devices are commonly used to qualify surface crystalline materials. However, this method is not convenient for disordered materials because of high defects density at insulator/material interface. In this study, surface quality of polycrystalline silicon thin films is investigated, based on the surface state theoretical model of Suzuki, and using temperature measurements of field effect conductance. Results show a higher surface state density for FSPC polycrystalline silicon TFTs. This is explained because the active