Tharindi Hapuarachchi
email: t.hapuarachchi@ucl.ac.uk

Chang Sub Park
email: chang.park@eng.ox.ac.uk

Stephen Payne
email: stephen.payne@eng.ox.ac.uk

Quantification of the effects of vasomotion on mass transport to tissue from axisymmetric blood vessels

Keywords: Oscillations, Ischemia, Non-linear dynamics

The process known as vasomotion, rhythmic oscillations in vessel diameter, has been proposed to act as a protective mechanism for tissue under conditions of reduced perfusion, since it is frequently only observed experimentally when perfusion levels are reduced. This could be due to a resultant increase in oxygen transport from the vasculature to the surrounding tissue, either directly or indirectly. It is thus potentially of significant clinical interest as a warning signal for ischemia. However, there has been little analysis performed to quantify the effects of vessel wall movement on time-averaged mass transport. We thus present a detailed analysis of such mass transport for an axisymmetric vessel with a periodically oscillating wall, by solving the non-linear mass transport equation, and quantify the differences between the time-averaged mass transport under conditions of no oscillation (i.e. the steady-state) and varying wall oscillation amplitude.

The results show that if the vessel wall alone is oscillated, with an invariant wall concentration, the time-averaged mass transport is reduced relative to the steady-state, but if the vessel wall concentration is also oscillated, then mass transport is increased, although this is generally only true when these oscillate in phase with each other. The influence of Péclet number and the non-

Introduction

Vasomotion is the process of oscillation in vascular tone, generated from within the vessel wall.

Although it has been seen in many studies over many years, the physiological consequences of vasomotion remain very unclear, [START_REF] Nilsson | Vasomotion: Mechanisms and physiological importance[END_REF]. One potential benefit is that the time-averaged hydraulic resistance to flow is lower than the steady state resistance, due to the non-linear relationship between vessel radius and resistance, [START_REF] Meyer | Reassessing the mathematical modelling of the contribution of vasomotion to vascular resistance[END_REF] Another hypothesis is that it improves tissue oxygenation, Tsai and Intaglietta, 1993, although this is controversial, [START_REF] Goldman | A computational study of the effect of vasomotion on oxygen transport from capillary networks[END_REF]. Experimental studies have shown that as perfusion levels are reduced, vasomotion is induced in most muscle preparations, Rücker et al., 2000. There is thus the possibility that vasomotion is induced under conditions of reduced perfusion as a protective mechanism against ischemia.

The equations governing mass transport to tissue, outlined below, are strongly non-linear (as is the equation relating flow to vessel diameter). A temporal oscillation of the vessel diameter about its steady-state value will cause the time-averaged behaviour to differ from the steadystate behaviour, possibly quite significantly at high oscillation amplitudes. However, solutions to these equations thus have to be considered carefully to ensure that the oscillating concentration profile in the tissue is characterised accurately. We present below a novel technique to solve for this concentration profile in the full non-linear case for a periodically oscillating wall diameter. This enables us to calculate the unsteady mass transport and hence to quantify the effects of vasomotion on the time-averaged mass transfer from individual vessels. We then conclude by 2 Theory

Concentration equation

We start from the general mass transport equation for the concentration of gas in the tissue surrounding a blood vessel:

where C is the concentration of gas dissolved in the tissue, U is the velocity field of the tissue, D is the diffusion coefficient of gas in the tissue and R is the rate of reaction (assumed negative here since the gas in question here, oxygen, is metabolised and hence essentially continuously removed from the tissue). For an axisymmetric vessel in cylindrical co-ordinates, assuming a rate of reaction linearly proportional to the concentration of gas in the tissue, this becomes:

where r is the radial coordinate measured from the centre of the vessel, R is the radius of the vessel (explicitly given as a function of time) and k is the metabolic rate constant. The assumptions made here will be discussed in full detail below in the context of the results. Note that the second term in equation 2, which introduces the non-linearity into the model, is derived from continuity considerations for the tissue surrounding the vessel.

Equation 2 is then re-cast in non-dimensional form, with respect to a length scale R 0 , a timescale T and the angular frequency of oscillation ω: where Pe = R 0 2 /DT is the Péclet number and , without loss of generality.

Using a similar approach to that of Fyrillas and Szeri, 1994, equation 4 is re-written in Lagrangian coordinates, where :

with boundary conditions:

where is the (possibly temporally varying) concentration of gas in the liquid at the wall. This is of course governed by the flow and transport conditions inside the vessel, but for now is specified independently. The second boundary condition assumes an infinitely far off boundary, which assumes that the vessel being modelled is independent of all other vessels. The likely impact of this on the solution will be discussed later. Note that the consumption rate constant is assumed to be invariant with space, without an explicit separate treatment of the vessel wall: this is based on the findings of Sharan et al., 2008, where the metabolic rate of oxygen was shown to be the same in the vessel wall as for the surrounding tissue.

A periodically oscillating boundary displacement is imposed: transients have died away, the gas concentration at each point in space will also be periodic in nature. This allows a separation of variables to be made for the concentration profile:

where and are complex conjugates. Note that a full Fourier series is used here to arbitrary order of n, as is required due to the non-linear nature of the governing equations. Such a series could also be used for the wall displacement if required, although only the lowest harmonics are likely to be significant in either expansion.

Substituting the above in equation ( 5) gives:

The terms in equation ( 9) corresponding to the 0 th and 1 st harmonics must individually balance.

We assume at this point that the 2 nd harmonic terms are negligible in comparison with the 0 th and 1 st harmonics, since the process is being driven by a wall movement containing only a fundamental component. It would, of course, be possible to map out all the harmonic terms in the solution by balancing up to arbitrary order in equation 9 but we do not do this here. Balancing the 0 th , 1 st and -1 st harmonic terms thus gives: 10), ( 11) and ( 12) then reduce to:

The complexity of these three coupled equations prevents an analytical solution from being derived. The presence of an infinitely far away boundary then also makes imposing suitable boundary conditions numerically difficult. To overcome this problem by converting the problem into a bounded co-ordinate system, equations ( 13), ( 14) and ( 15) are re-written using the coordinate transformation :

Note that the problem now reduces to 3 simple 2 nd order differential equations that can easily be solved numerically within the range (s = 0 corresponding to the tissue infinitely far Note that in the steady state, equation ( 16) reduces to:

which can again easily be solved with the same boundary conditions, enabling a direct comparison to be made with the oscillating solution.

Mass transport equation

Before examining the solutions to the concentration equations, we examine the mass transport equation, since the aim is to compare the rate of mass transfer from vessel to tissue under oscillating and non-oscillating conditions. The rate of diffusive mass transport of gas across the wall is given by: where is the diffusion coefficient of gas through the wall. In Lagrangian coordinates:

Substitution of equations ( 7) and ( 8) and transformation into the bounded coordinate system yields:

for the time averaged mass transport. Note that the oscillating mass transport component is ignored here since it makes no contribution to the net mass transport from blood to tissue. In the steady state the rate of mass transport is given by: where this is calculated directly from the solution to equation ( 19). Note that the expression for is not the same as the expression for in equation ( 22). This is due to the nonlinearities in the system affecting the time-averaged mass transport as described previously. The ratio of time-averaged mass transport to steady state mass transport is then:

which we term the Mass Transport Ratio. It is this ratio that is of primary interest here.

Results

Two sets of solutions are considered here. In the first set, the wall is oscillated but the wall concentration is kept constant. The boundary conditions thus become:

In the second, both the wall and the wall concentration are oscillated. The boundary conditions then become:

A c c e p t e d m a n u s c r i p t 9 and are set by the magnitude and phase of the wall concentration oscillations, relative to the wall displacement. For all the simulations presented below, the governing equations were solved with the specified boundary conditions using the Matlab solver, bvp4c. From the complete solution for the concentration profile, the wall concentration gradients in equation ( 24) were then estimated numerically.

Since the absolute phase of the solutions is arbitrary, we set b=0 without loss of generality.

Equation ( 7) then simplifies to . The wall displacement oscillations are thus defined to be the reference phase, relative to which are measured the concentration oscillations.

Note that the results are only dependent on the values of two non-dimensional parameters (Péclet number and non-dimensional rate constant) and the amplitude of the wall displacement (2a) and amplitude and phase of the wall concentration ( and ) oscillations.

To illustrate the model predictions, the results for , and are shown against the coordinate s for the condition where K = 1, a = 0.2 and Pe = 0.1 in Figure 1. Despite the fact that the oscillating components are constrained to be zero at both ends of the co-ordinate system and to have zero gradient at the outer boundary (s = 0), there is a non-zero gradient at the inner boundary (s = 1), which is what alters the time-averaged mass transport from that in the steady state (equation 24). Note that the gradient for is negative, which is why the time-averaged mass transport is lower than in the steady state.

Wall displacement oscillation only

We first consider the effects of oscillating only the wall displacement. Figure 2 shows the effect of vasomotion on the Mass Transport Ratio at different values of the Péclet number for a fixed value of K = 1 and a range of wall amplitudes, a < 0.5. For all solutions, as the wall amplitude tends towards zero, the mass transfer ratio tends towards 1, as expected, since the governing equations For an increased value of K = 10, the results are very similar, Figure 3. The mass transfer ratio is slightly higher than for K = 1, but the difference is only slight. For a decreased value of K, as shown in Figure 4, the results are likewise rather insensitive to the precise value of K apart from at high values of Péclet number, where there is a slight increase in the time-averaged mass transport.

The mass transport ratio, however, is uniformly less than 1 for all the values considered here, indicating that the oscillation in wall displacement causes a reduction in mass transfer over all ranges of parameter values investigated.

Wall displacement and wall concentration oscillations

We next consider the case where the wall concentration also oscillates. Four cases are shown in The interaction between wall displacement and wall concentration oscillations is clearly substantially more complicated than for wall displacement oscillations alone. Under most conditions, the mass transport reduces, but there are some cases when this mass transport increases, potentially very significantly.

When the wall displacement and wall concentration oscillations are in phase, at Péclet numbers greater than about 1, the mass transport is found to increase over all values of a. However, for Péclet numbers smaller than this, the ratio is less than 1, indicating a drop in mass transport.

Discussion

The results presented above show that the time-averaged mass transport is very significantly different from the steady-state mass transport under a wide range of conditions. We now consider the typical values of the non-dimensional parameters found in the vasculature before examining the accuracy and potential physiological implications of the results obtained here.

The diffusion coefficient of oxygen in tissue, D, has been measured at 37 °C to be 2.4x10 -9 m 2 /s [START_REF] Bentley | Temperature dependence of oxygen diffusion and consumption in mammalian striated muscle[END_REF] and this value is used throughout. The metabolic rate constant is more complicated, as the linear consumption equation used here is only an approximation to the complex underlying metabolic processes. We provide a first order estimate by dividing the metabolic rate of oxygen by the typical concentration of oxygen in the tissue. The former is quoted as 8.2x10 -4 cm 3 O 2 /cm 3 /s and the latter as 2.6x10 -4 cm 3 O 2 /cm 3 [START_REF] Mintun | Blood flow and oxygen delivery to human brain during functional activity: Theoretical modelling and experimental data[END_REF], yielding an approximate value for the rate constant of 3.2 /s. This is clearly a gross estimate for this parameter, but the fact that it is greater than 1 indicates (from the results presented earlier) that there is little sensitivity to its precise value. We note in passing that spatial measurements of

A c c e p t e d m a n u s c r i p t

12 static oxygen partial pressure in tissue remain highly controversial and uncertain, see for example [START_REF] Golub | PO 2 profiles near arterioles and tissue oxygen consumption in rat mesentery[END_REF]Tsai et al., 1998, which Geometrically it has been assumed that the vessel is axisymmetric in an infinite tissue. The assumption of an infinite volume of tissue surrounding each vessel is clearly inaccurate and is included here to reduce the number of parameters involved in the model. A Krogh style cylinder is often used in similar models, but the results can be sensitive to the particular choice of outer radius, which makes interpretation of the results difficult. However, the primary reason for choosing an infinite volume is the fact that the governing equation used here is linear in concentration. The actual tissue concentration can then be considered simply as the summation of the solutions for all individual vessels, each of which is surrounded by an infinite volume of tissue. Although this will have some inherent inaccuracies, as long as the vessels are reasonably well-spaced, it is likely that the mass transport from individual vessels will be well captured.

The other major assumption made here is that there are no axial variations in the concentration or displacement profiles, this being done since it reduces the computational requirement very substantially. We can however, estimate their effects by considering the effects of axial waves along the vessel (the possible cause of such waves will be considered later). The governing equation (2) becomes:

where z is the axial coordinate. In non-dimensional form, this becomes: where the non-dimensional wavelength of the axial waves is , this depending on the imposed form of the solution. The equivalent form of equation ( 9) is then:

The only effect of imposing an axial wave-like behaviour is thus to increase the effective value of the non-dimensional metabolic rate constant from K to . The general solution to equation 30 is otherwise unaffected. The effect of axial variations will thus depend upon both the absolute change in the effective value of K and the sensitivity of the solution to these changes. If it is assumed that the non-dimensional wavelength is of order 1 (i.e. the wavelength is the same order of magnitude as the vessel length) and that the vessel radius to length ratio is of order 0.1 (since most vessels are significantly longer than their radius), the increase in effective K is of order 0.01/Pe. This will be greatest when the Péclet number is smallest, which occurs in the smallest vessels, where it is approximately 0.02, yielding an increase in effective K of less than 1. Since baseline K is around 3 and the results shown previously indicate that above a value of approximately 1, the precise value of K has little impact on the results, axial variations are likely to have only a second order impact on the mass transport.

Clearly, however, the model presented here has significant limitations in the assumption of a linear model for concentration and metabolism. This was done here simply to gain an insight into the previously unconsidered process of the dynamic interaction between wall displacement and Although the values of model parameters can be estimated reasonably accurately from the experimental literature, direct validation of the results presented here is extremely difficult.

Measurement of the dynamic oxygen concentration profile surrounding a vessel to the required order of accuracy is experimentally very challenging and no suitable data exist to the best of our knowledge. Measurement of the changes in time-averaged mass transport would appear only to be experimentally feasible by quantifying the changes in oxygen saturation of the blood exiting a vasomotive vessel or network to test whether this is higher under vasomotive conditions than under normal conditions. However, since vasomotion is normally induced by a reduction in perfusion, which will also directly lead to a substantive change in the oxygen saturation of the exiting blood, it is debatable whether such a reduction is capable of being measured accurately enough to provide a direct validation of the results presented in this paper.

There has been some debate in the literature about the role of vasomotion in promoting the supply of oxygen to tissue, particularly under conditions of reduced perfusion, when the supply would otherwise be expected to be reduced with severe consequences. The role of vasomotion as a protective mechanism was first proposed by Tsai and Intaglietta, 1993, where it was suggested that long-range oxygen diffusion was increased under such conditions, thus providing a protective mechanism for vulnerable tissue. This was re-examined by [START_REF] Goldman | A computational study of the effect of vasomotion on oxygen transport from capillary networks[END_REF], where it was concluded that this was only the case at low myoglobin levels. Rucker et al., 2000, found experimentally that vasomotion promoted the supply of blood to adjacent tissues, which implies

A c c e p t e d m a n u s c r i p t

16 that there are additional processes occurring during the conditions that induce vasomotion, which may not be directly related to the vasomotive process.

The vascular network is highly complex with vessels over a wide range of length scales and active autoregulatory processes affecting vessel diameters across the network (particularly in the arteriolar bed) when perfusion levels drop. Disentangling all of the different effects that occur under conditions of reduced perfusion is thus a substantial task, especially when attempting to interpret experimental data. This complexity is undoubtedly one of the key reasons why "the conclusions borne from theoretical studies have received little direct support from experiments", [START_REF] Nilsson | Vasomotion: Mechanisms and physiological importance[END_REF]. The model that we present here thus attempts to shed additional insight onto this process by considering a previously neglected component, i.e. the non-linear interaction between the vessel wall movement and the dynamic concentration field in the surrounding tissue. We have neglected many other components of the problem, which will need to be examined in detail, together with the dynamic interaction considered here, to gain a full insight into the potentially key role of vasomotion in tissue protection. In particular, the nonlinear dynamics of oxygen within tissue are likely to play a key role.

However, the results presented here do provide some interesting possibilities. For the typical values of Péclet number and non-dimensional rate constant calculated here, mass transport is reduced under nearly all conditions (except when the wall concentration oscillations are at a certain phase relative to the wall displacement oscillations, and even then the increase is very small). This reduction in the time-averaged mass transport between the oscillating and nonoscillating conditions appears rather surprising, given the discussion in the literature about vasomotion being a potential protective mechanism under conditions of ischemia. However, this process has to be considered within a complete network of vessels.

A c c e p t e d m a n u s c r i p t 17

The behaviour of such a network in the context of vasomotion has previously been considered by, amongst others, [START_REF] Goldman | A computational study of the effect of vasomotion on oxygen transport from capillary networks[END_REF]Tsoukias et al., 2007. The former found that at physiological levels of tissue myoglobin vasomotion did not improve tissue oxygenation (but did so under other conditions). However, neither model considered the physical movement of the vessel walls as playing an important part in the tissue oxygenation process: only the flow field was oscillated and only the capillary bed was considered. This is similar to the work of Schneiderman et al., 1982, which examined the effects of oscillations in flow only. We have extended this work by considering the larger arteriolar vessels where vasomotion occurs, but have only considered a single vessel: more analysis will be required to investigate these vessels in a similar manner to these previous studies.

Oxygen delivery to tissue is found at both the arteriolar and capillary level, as shown by experimental data recorded by Vovenko, 1999, where the partial pressure of oxygen at inlet to the capillary bed was only of order 60 mmHg. The transport of oxygen to tissue thus occurs over a range of length scales, making interpreting the net effect of vasomotion highly complicated.

Vasomotive activity in the arteriolar bed appears to lead to a reduction in oxygen delivery to tissue: however, this ensures a greater oxygen concentration at entrance to the capillary bed. This would appear likely to promote oxygen transport within the capillary bed and thus could help to explain why vasomotion acts as a protective mechanism at the capillary level, rather than at the arteriolar level. It would then be seen as a means of retention of more oxygen for supply to the capillary bed.

If oscillations are present in the arteriolar vessel walls, this will also have the effect of perturbing the capillary flow field, both in terms of flow and concentration. It is not clear whether these oscillations will remain in significant amplitude at the capillary bed, since the arterial network acts as a low-pass filter to reduce the amplitude of flow oscillations (although significant oscillations in A c c e p t e d m a n u s c r i p t 18 red blood cell velocity have been observed experimentally in capillaries, [START_REF] Ellis | Effect of a maldistribution of microvascular blood flow on capillary O2 extraction in sepsis[END_REF], and, if they do, whether they would be sufficiently large to promote oxygen delivery in individual vessels in addition to the fact that the time-averaged oxygenation would also be higher. Much future work will be required to investigate the effects of this phenomenon across the vasculature.

In particular, the relationship between perturbations in the wall displacement and wall concentration will need to be determined. This could be done by solving the equations of motion and mass transport within the vessel and coupling them to the equations for wall displacement.

Although this is relatively complicated, it would determine more definitely the nature of this coupling, which has been taken to be unknown here, and thus aid in interpreting the underlying physiological basis of vasomotion.

Conclusions

In this paper we have presented a technique for solving the non-linear mass transport equation for axisymmetric, axially invariant unbounded vessel-tissue systems with a periodically oscillating boundary. The results show that vasomotion alone causes the time-averaged mass transport to decrease under virtually all conditions, but that if the wall concentration oscillates in phase with this movement, then the time-averaged mass transport can increase under certain conditions.

We therefore hypothesise that vasomotion might occur in the arteriolar network to decrease oxygen supply at this vascular level in order to increase the transport of oxygen to the capillary bed, which could have protective implications under conditions of reduced perfusion and thus explain why vasomotion plays a role under such conditions.

  implications of this for our understanding of the physiological role of vasomotion.

  conjugates, the magnitude of which are proportional to the amplitude of oscillations. Given the periodic nature of the wall movement, once any initial

  solution, we re-write the coefficients in real and imaginary parts, such that where a and b govern the magnitude and phase of the imposed boundary oscillation. Equations (

  vessel and s = 1 corresponding to the vessel wall). The boundary conditions are set by the particular nature of the problem under investigation, as discussed below.

  the limit. As a increases, however, this ratio decreases monotonically: at low amplitudes of oscillation, the effect of Péclet number is small, but at amplitudes greater than approximately a > 0.25, decreases in Péclet number result in a reduction in mass transfer ratio.

Figures 5

 5 Figures 5-8, considering the cases where the amplitude of concentration oscillation is 0.5: Figure

  oscillates 90 degrees ahead, the mass transport is lower under virtually all combinations of Péclet number and displacement amplitude: only for moderate amplitudes and very low values of Péclet number is the mass transport enhanced, and this is a small effect. Similar results are found for a 180 degree phase shift: only at moderate amplitudes of displacement oscillation and very low Péclet number does the mass transport increase, and again only fractionally. The results for a 90 degrees phase shift behind, however, are quite substantially different. For low values of Péclet number, the mass transport decreases over all values of a, but for values greater than approximately Pe = 1, there is a moderate increase in mass transport over a wide range of a.

  -dimensional axial coordinate and is the characteristic length of the vessel. The trial solution for the concentration profile would be of the form:

  by taking the form of the equation that provides a physiologically realistic concentration profile in the simplest mathematical form. It will be necessary in future to consider a more detailed model for oxygen tension and myoglobin saturation (as done inGoldman and Popel, 2001, for example) and to attempt to validate the static concentration field against experimental data, although this remains challenging, as previously discussed.

Figure 1 -Figure 3 -Figure 4 -Figure 5 -Figure 7 -Figure 8 -Figure 1 -

 1345781 Figure Captions

Figure 2 -Figure 3 -Figure 4 -Figure 5 -

 2345 Figure 2 -The effect of vasomotion on the rate of mass transport at different values of the Péclet number, at K=1.

Figure 6 -

 6 Figure 6 -The effect of vasomotion on the rate of mass transport at different values of the Péclet number, with concentration oscillating 90° in advance of the wall.

Figure 7 -

 7 Figure 7 -The effect of vasomotion on the rate of mass transport at different values of the Péclet number, with concentration oscillating 180° in advance of the wall.

Figure 8 -

 8 Figure 8 -The effect of vasomotion on the rate of mass transport at different values of the Péclet number, with concentration oscillating 270° in advance of the wall.

Table 1

 1 makes evaluation of the metabolic rate constant from experimental data still difficult.Colantuoni et al., 1984, measured the vasomotive activity in hamsters using image analysis techniques to capture the frequency and amplitude of oscillation of four types of arteriolar vessels (mean arteries, small arteries, arterioles and terminal arterioles). They found that the amplitude of oscillation increased with vessel radius as well as with frequency. The resulting values of Péclet number and non-dimensional rate constant are given in Table1. Note that the Péclet number varies over nearly two orders of magnitude over the arterial network, since it is proportional to the square of the baseline vessel radius. K varies over a much smaller range, since the oscillation frequency is nearly invariance with radius. In the smallest vessels, vasomotion results in very large displacement amplitudes. Parameter values and non-dimensional quantities taken from[START_REF] Colantuoni | Quantitation of rhythmic diameter changes in arterial microcirculation[END_REF]

	Type of	Mean radius	Oscillation frequency	Amplitude of
					Pe	K
	vessel	(µm)	(cycles/min)	oscillation
	Mean artery 85	2.74	19.3 %	0.864	11.2
	Small artery 55.7	3.36	20.8 %	0.455	9.09

Rucker et al., 2000

, measured the vasomotive activity in rats in both the transverse and terminal arterioles, with their experimental values yielding a Péclet number of approximately 0.02 (based on a diameter of 10 µm) and K of approximately 15. The amplitude of oscillation was found to be approximately 60 %, these values being in fairly good agreement with those of

[START_REF] Colantuoni | Quantitation of rhythmic diameter changes in arterial microcirculation[END_REF]

(although the frequency of oscillation was found to be somewhat slower). Although these measurements were not made in humans, they are in good agreement for Péclet number with only a small (and irrelevant) difference in K.

Acknowledgments

This work was supported by a grant from the Wellcome Trust for a Biomedical Vacation Scholarship for TH.

1 This work was performed in Oxford as part of a Wellcome Trust Biomedical Vacation Scholarship.


Stephen Todd
email: s.todd@qub.ac.uk

Bernadette Mcguinness

Peter Passmore

A Peter Passmore

Prevention of dementia by ACE inhibitors and angiotensin receptor blockers -potential but not proven

Dementia currently exacts a substantial economic burden on societies, health and social care systems, and carers in addition to the devastating personal and emotional cost to patients and families [1,2]. This burden is predicted to increase substantially in the coming decades and is the major public health issue of this century [1]. Currently available therapies are symptomatic only [3], with little or no prospect of disease-modifying treatments entering routine clinical use in the short to medium term. Strategies to prevent or delay the onset of dementia or retard its' progression are, therefore, urgently required and will be of extreme importance to individuals and societies. Currently available medications that could achieve these goals would have many advantages -potential for immediate use; established safety profile; familiarity to clinicians; relatively low cost giving favourable cost-effectiveness breakdown.

Cardiovascular risk factors and dementia

Recent reports have highlighted that a number of common cardiovascular risk factors are associated with both Alzheimer's disease (AD) and vascular dementia (VaD), the two most prevalent forms of dementia [START_REF] Qiu | The age-dependent relation of blood pressure to cognitive function and dementia[END_REF][START_REF] Kivipelto | Cholesterol as a risk factor for Alzheimer's diseaseepidemiological evidence[END_REF]. Additionally, post mortem studies have demonstrated that the characteristic neuropathological changes of both AD (senile plaques and neurofibrillary tangles) and VaD (subcortical/white matter ischaemia) are highly prevalent in the brains of normal individuals as well as those with AD, VaD, and other forms of dementia [START_REF] Schneider | Mixed brain pathologies account for most dementia cases in community-dwelling older persons[END_REF][START_REF] Schneider | The Neuropathology of Older Persons with and Without Dementia from Community versus Clinic Cohorts[END_REF][START_REF] Matthews | Epidemiological pathology of dementia: attributable-risks at death in the Medical Research Council Cognitive Function and Ageing Study[END_REF].

From this and earlier work, the hypothesis has arisen that treatment of these common cardiovascular risk factors, in particular hypertension and hypercholesterolaemia, will result in a reduction in the incidence and/or progression of dementia. Our group has performed systematic reviews and meta-analyses of blood pressure lowering therapies [START_REF] Mcguinness | Blood pressure lowering in patients without prior cerebrovascular disease for prevention of cognitive impairment and dementia[END_REF] and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin) treatment [START_REF] Mcguinness | Statins for the prevention of dementia[END_REF] for the prevention of dementia and/or cognitive impairment. At present, there is no evidence from randomised clinical trials (RCTs) that treatment of either hypertension or hypercholesterolaemia reduces the incidence of dementia [START_REF] Mcguinness | Blood pressure lowering in patients without prior cerebrovascular disease for prevention of cognitive impairment and dementia[END_REF][START_REF] Mcguinness | Statins for the prevention of dementia[END_REF]. However, we found several limitations in the literature.

In the blood pressure lowering systematic review [START_REF] Mcguinness | Blood pressure lowering in patients without prior cerebrovascular disease for prevention of cognitive impairment and dementia[END_REF], significant bias may have been introduced by the number of participants lost to follow up, with differential loss between active treatment and placebo groups, and the large proportions of placebo allocated participants receiving active treatment in the included RCTs. The primary outcomes in the included studies were cardiovascular and the studies stopped when significant benefits in these primary outcomes were found. Dementia or cognitive impairment were secondary outcomes and the assessments used, the duration of the trials, and the number of participants randomised may have been insufficient to have detected true significance for these outcomes. In the statin prevention systematic review [START_REF] Mcguinness | Statins for the prevention of dementia[END_REF], only two studies met the inclusion criteria and, again, cognition was a secondary or tertiary outcome. There was a low rate of incident dementia in both placebo and active treatment groups.

Do ACE inhibitors and angiotensin type 2 receptor blockers reduce incidence or progression of dementia?

Two analyses from prospective cohort studies have been reported recently that add further data to the debate on treatment of hypertension for benefits on cognition [11, . Sink and colleagues [START_REF] Sink | Angiotensin-converting enzyme inhibitors and cognitive decline in older adults with hypertension: results from the cardiovascular health study[END_REF] examined data from participants of the Cognition substudy of the Cardiovascular Health Study [START_REF] Lopez | Evaluation of dementia in the cardiovascular health cognition study[END_REF] on the effect of ACEI use on cognitive decline. ACEIs were classified into those that crossed the BBB (centrally active) and those that did not (non-centrally active) using data mainly from animal studies [START_REF] Sink | Angiotensin-converting enzyme inhibitors and cognitive decline in older adults with hypertension: results from the cardiovascular health study[END_REF]. The majority of the 1054 participants in the study were female (64%) and white (76%), with a mean age of 75 years. ACEIs were not associated with a reduction in the risk of dementia when compared as a class to other antihypertensive drug classes (adjusted HR 1.01; 95% CI 0.87-1.18). However, centrally active ACEIs were associated with a 65% reduction in cognitive decline, as measured by the Modified Mini-Mental State Examination (3MSE) [START_REF] Teng | The Modified Mini-Mental State (3MS) examination[END_REF], per year of drug exposure as compared to other anti-hypertensive medications (p=0.01). Noncentrally active ACEIs were associated with a 73% increased risk of dementia over three years, compared with other anti-hypertensive agents.

The brain, as with most organs, has a paracrine renin-angiotensin system (RAS). In brain, RAS has specific local effects, including on memory, independent of the systemic circulatory RAS, unlike other organs such as the heart or kidney [START_REF] Fyhrquist | Renin-angiotensin system revisited[END_REF].

Preliminary data suggest possible effects of drugs acting on RAS on cognition beyond blood pressure (BP) control. ACE activity is increased in AD post mortem brain and correlates with amyloid plaque load and with AD severity (reviewed in [START_REF] Kehoe | Angiotensins in Alzheimer's disease -friend or foe?[END_REF]). Some, but not all, animal studies show alteration in amyloid pathology and cognitive performance with RAS active medications (reviewed in [START_REF] Kehoe | Angiotensins in Alzheimer's disease -friend or foe?[END_REF]). ACE degrades amyloid-β (Aβ) peptide, the precursor of plaques, in the brain and captopril caused an increase in the neurotoxic Aβ 42 in one mouse model [START_REF] Zou | Angiotensin-converting enzyme converts amyloid beta-protein 1-42 (Abeta(1-42)) to Abeta(1-40), and its inhibition enhances brain Abeta deposition[END_REF]. Therefore, will long term ACEI therapy have detrimental effects on cognition? Various mouse [START_REF] Kehoe | Angiotensins in Alzheimer's disease -friend or foe?[END_REF][START_REF] Wang | Valsartan lowers brain beta-amyloid protein levels and improves spatial learning in a mouse model of Alzheimer disease[END_REF][START_REF] Takeda | Angiotensin receptor blocker prevented betaamyloid-induced cognitive impairment associated with recovery of neurovascular coupling[END_REF]. However, it may not be appropriate to extrapolate animal models to humans. Other small observational studies have also reported reduced progression of mild cognitive impairment to AD [START_REF] Rozzini | Angiotensin converting enzyme (ACE) inhibitors modulate the rate of progression of amnestic mild cognitive impairment[END_REF] and reduced incidence of AD [START_REF] Ohrui | Angiotensin-converting enzyme inhibitors and incidence of Alzheimer's disease in Japan[END_REF]. One small RCT has suggested that centrally active ACEIs slow cognitive decline in AD more than non-centrally active ACEIs or calcium channel blockers [START_REF] Ohrui | Effects of brain-penetrating ACE inhibitors on Alzheimer disease progression[END_REF].

Li and colleagues reported that ARBs were associated with a reduction in the incidence and progression of dementia and AD compared with the ACEI lisinopril and with "cardiovascular comparators" [START_REF] Li | Use of angiotensin receptor blockers and risk of dementia in a predominantly male population: prospective cohort analysis[END_REF]. Of note, Li classified lisinopril as noncentrally active, whereas in Sink's study lisinopril was classified as centrally active. Li et al. based their classification on the hydrophilicity of lisinopril [START_REF] Ranadive | Relative lipophilicities and structuralpharmacological considerations of various angiotensin-converting enzyme (ACE) inhibitors[END_REF]. However, this report references an animal study showing prolonged cerebrospinal fluid ACE inhibitory activity by lisinopril [START_REF] Cushman | Differentiation of angiotensin-converting enzyme (ACE) inhibitors by their selective inhibition of ACE in physiologically important target organs[END_REF], suggesting a possible peptide carrier at the BBB.

Sink classified central activity or non-activity on the basis of this earlier study [START_REF] Sink | Angiotensin-converting enzyme inhibitors and cognitive decline in older adults with hypertension: results from the cardiovascular health study[END_REF].

The summary of product characteristics for lisinopril states "studies in rats indicate lisinopril crosses the BBB poorly". The cohort was predominantly male (98%) with a mean age of 74 years. Ethnicity was unknown for most subjects. There are several limitations of this study. Firstly, the three groups are not comparable as there are significant differences in morbidity profiles and mean BP and small differences in ethnicity. All of these impacts on the risk or recognition of dementia. The "cardiovascular comparator" group had lower rates of hypertension and diabetes and higher rates of cardiovascular disease and stroke than the ARB and lisinopril groups.

Mean BP was 3/2mmHg lower in the "cardiovascular comparator" group. Low BP in late-life is associated with dementia and AD [START_REF] Qiu | The age-dependent relation of blood pressure to cognitive function and dementia[END_REF]. The level of absolute BP reduction rather than the agent(s) used is considered paramount in reduction of cardiovascular mortality and morbidity. Secondly, the prevalence of use of other cardiovascular medications, such as anti-platelet or other anti-hypertensive agents, across the three groups was not reported. Statins were specifically excluded from the "cardiovascular comparator" group, with no rationale provided. The corresponding author had previously reported a similar large association between use of statins and decreased prevalence of AD [START_REF] Wolozin | Decreased prevalence of Alzheimer disease associated with 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitors[END_REF]. However, subsequent RCTs have not shown a beneficial effect of statins on the prevention of dementia [START_REF] Mcguinness | Statins for the prevention of dementia[END_REF]. The progression outcome (nursing home admission) was primarily driven by reduction in stroke in the ARB group. Modification of risk factors other than BP by use of other agents may have contributed to this. However, in a RCT of an ACEI and thiazide diuretic regimen, reduction in stroke recurrence was associated with reduced dementia incidence [START_REF] Tzourio | Effects of blood pressure lowering with perindopril and indapamide therapy on dementia and cognitive decline in patients with cerebrovascular disease[END_REF].

Finally, the clinical plausibility of such large reductions in the incidence and progression of dementia and AD in a late-life population with relatively short follow up was not considered. As noted earlier, AD and vascular neuropathological changes are highly prevalent in this age-group [START_REF] Schneider | The Neuropathology of Older Persons with and Without Dementia from Community versus Clinic Cohorts[END_REF] and have been present for many years before incident dementia becomes symptomatic and is diagnosed.

Vascular risk factor interventions in dementia

There are few interventional studies of vascular risk factor modification, either singly or in combination, in AD. Observational studies suggested cognition declined less on statin treatment [START_REF] Masse | Lipid lowering agents are associated with a slower cognitive decline in Alzheimer's disease[END_REF] and function declined less on statin and β-blocker treatment An observational study reported that treatment of vascular risk factors was associated with slower cognitive decline in patients with AD and no CVD than no treatment [START_REF] Deschaintre | Treatment of vascular risk factors is associated with slower decline in Alzheimer disease[END_REF]. One recent small RCT failed to show a benefit of intensive vascular risk factor modification over usual care in slowing dementia progression in subjects with AD and vascular lesions on neuroimaging [START_REF] Richard | Evaluation of Vascular care in Alzheimer's disease. Vascular care in patients with Alzheimer's disease with cerebrovascular lesions-a randomized clinical trial[END_REF]. It may be that this stage of the disease process with established neuropathology is too late to initiate these treatments. However, although BP was reduced in both intensive and usual care groups, the difference was not significant [START_REF] Richard | Evaluation of Vascular care in Alzheimer's disease. Vascular care in patients with Alzheimer's disease with cerebrovascular lesions-a randomized clinical trial[END_REF]. It is possible that lowering BP and cholesterol in AD may be harmful as blood pressure falls in older people with AD [START_REF] Qiu | The age-dependent relation of blood pressure to cognitive function and dementia[END_REF] and cholesterol may be lower in AD [START_REF] Kivipelto | Cholesterol as a risk factor for Alzheimer's diseaseepidemiological evidence[END_REF]. A trial is ongoing to establish if a similar regimen is of benefit in reducing dementia incidence in elderly people with vascular risk factors (ISRCTN29711771) [START_REF] Richard | Prevention of dementia by intensive vascular care (PreDIVA): a cluster-randomized trial in progress[END_REF].

Conclusions

Given the current lack of reliable RCT evidence and these interesting but limited retrospective, observational studies, several questions remain to be answered: does treatment of hypertension in mid-life result in reduced risk of dementia or cognitive decline in late-life? Does treatment of hypertension in late-life result in reduced incidence and/or progression of dementia? If so, is one anti-hypertensive class, or sub-group that crosses the BBB, associated with these better cognitive outcomes? Are certain anti-hypertensives, such as ACEIs discussed previously [START_REF] Kehoe | Angiotensins in Alzheimer's disease -friend or foe?[END_REF], associated with worse cognitive outcomes? Does addressing the overall cardiovascular risk with lifestyle interventions, antiplatelet and lipid lowering therapies, in addition to antihypertensives, provide additional benefit in reducing dementia incidence or progression? As has been suggested previously by other commentators [START_REF] Kehoe | Angiotensins in Alzheimer's disease -friend or foe?[END_REF][START_REF] Kehoe | Is inhibition of the renin-angiotensin system a new treatment option for Alzheimer's disease?[END_REF], the recent reports highlight that well designed RCTs, adequately powered and of sufficient duration to answer these questions, with appropriate primary outcomes which assess dementia (and subtype) incidence and progression are urgently required. The use of effective, inexpensive, currently available therapies to reduce the considerable burden from dementia would be of major benefit to older individuals and their carers, health and social systems, and economies not only in the developed world but also the developing world [START_REF] Ferri | Global prevalence of dementia: a Delphi consensus study[END_REF].

  ARBs have been associated with reduced Alzheimer's neuropathology and improved performance on tests of learning and memory

  . However, RCTs have not shown benefit of statins on progression of cognition in AD.

1

  article, critical revision of article, approval of article BMcG: critical revision of article, approval of article APP: critical revision of article, approval of article Knapp M, Prince M, Albanese E, Banerjee S, Dhanasiri S. Dementia UK: A report into the prevalence and cost of dementia prepared by the Personal Social Services Research Unit (PSSRU) at the London School of Economics and the Institute of Psychiatry at King's College London, for the Alzheimer's Society. London: Alzheimer's Society, 2007. 2 National Collaborating Centre for Mental Health. Dementia: a NICE-SCIE guideline on supporting people with dementia and their carers in health and social care. London: Royal Psychological Society, 2007. 3 Birks J. Cholinesterase inhibitors for Alzheimer's disease. Cochrane Database Syst


Sónia Ferreira

Mark Toleman

Elmano Ramalheira

Gabriela J Da Silva

Timothy Walsh

Sónia Mendo
email: smendo@ua.pt

First description of Klebsiella pneumoniae clinical isolates carrying both qnrA and qnrB genes in Portugal 

Keywords:

A c c e p t e d M a n u s c r i p t

Introduction

Plasmid-mediated quinolone resistance encoded by qnrA, qnrB or qnrS genes has been reported worldwide [START_REF] Nordmann | Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae[END_REF][START_REF] Wang | Occurrence of qnr-positive clinical isolates in Klebsiella pneumoniae producing ESBL or AmpC-type -lactamase from five pediatric hospitals in China[END_REF]. Qnr proteins are members of the pentapeptide repeat protein family that protect topoisomerases and thereby reduce their susceptibility to fluoroquinolones [START_REF] Nordmann | Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae[END_REF][START_REF] Cavaco | First detection of plasmid-mediated quinolone resistance (qnrA and qnrS) in Escherichia coli strains isolated from humans in Scandinavia[END_REF]. qnrA was the first qnr gene described in a Klebsiella pneumoniae in 1998 by Martínez-Martínez et al. [START_REF] Martínez-Martínez | Quinolone resistance from a transferable plasmid[END_REF]. Subsequently, it has been shown to be widely spread worldwide as part of a complex sul1-type integron containing ISCR1 [START_REF] Nordmann | Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae[END_REF][START_REF] Park | Prevalence and diversity of qnr alleles in AmpC-producing Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii and Serratia marcescens: a multicentre study from Korea[END_REF]. qnrB and qnrS have also been reported in many countries from Europe, Asia and the USA [START_REF] Nordmann | Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae[END_REF]. Dissemination of plasmids carrying these genes can potentiate the rapid development of higher-level quinolone resistance in bacterial strains currently classified as susceptible [START_REF] Robicsek | Broader distribution of plasmid-mediated quinolone resistance in the United States[END_REF]. Moreover, transferrable qnr genes are usually carried by conjugative plasmids (50-180 kb) that often encode extendedspectrum -lactamases (ESBLs) or AmpC-type -lactamases [START_REF] Lascols | A plasmid-borne Shewanella algae gene, qnrA3, and its possible transfer in vivo between Kluyvera ascorbata and Klebsiella pneumoniae[END_REF]. In fact, epidemiological studies of the distribution of qnr determinants show that qnrpositive strains frequently express ESBLs [START_REF] Garnier | Genetic environment of quinolone resistance gene qnrB2 in a complex sul1-type integron in the newly described Salmonella enterica serovar Keurmassar[END_REF]. Here we report the presence of qnr genes in quinolone-resistant K. pneumoniae and investigate their association with different -lactamases.

Material and methods

Klebsiella pneumoniae clinical isolates

Twenty-one K. pneumoniae were isolated from various biological products, including urine, wound swabs, respiratory secretions, blood and sterile body Pedro (Aveiro, Portugal). Species identification was performed using the VITEK 2 system (bioMérieux, Marcy l'Etoile, France). The population analysed was non-repetitive and only a single isolate per patient was included in the study.

Drug susceptibility testing

Susceptibility testing was performed according to the guidelines of the Clinical and Laboratory Standard Institute [START_REF]Performance standards for antimicrobial susceptibility testing[END_REF]. ESBL production was confirmed by Etest with cefotaxime/cefotaxime + clavulanic acid, ceftazidime/ceftazidime + clavulanic acid and cefepime/cefepime + clavulanic acid strips, according to manufacturer's instructions (AB BIODISK, Solna, Sweden).

Hybridisation assays

Resolution of S1-digested genomic DNA of K. pneumoniae isolates was performed with a CHEF-DRIII system (Bio-Rad, Hemel Hempstead, UK). PCR detection of class 1 integrons was performed as previously described [START_REF] Barlow | Isolation and characterization of integron-containing bacteria without antibiotic selection[END_REF]. The presence of qnr genes was detected by PCR using the primers described by Wu et al. [START_REF] Wu | Prevalence of plasmid-mediated quinolone resistance determinants QnrA, QnrB, and QnrS among clinical isolates of Enterobacter cloacae in a Taiwanese hospital[END_REF]. All isolates were screened by PCR for the presence of SHV, CTX-M, TEM and OXA -lactamases as well as AmpC groups (DHA, MOX, FOX, CMY and ACC) [START_REF] Henriques | Occurrence and diversity of integrons and -lactamase genes among ampicillin-resistant isolates from estuarine waters[END_REF]. Nucleotide sequences of PCR products were determined and compared with other sequences deposited in the EMBL GenBank.

Conjugation assays

Conjugation assays were performed with Escherichia coli J53 Azi r (azideresistant) as recipient and KP4, KP5, KP6 and KP10 as donors. Strains were routinely grown in Luria-Bertani broth. Culture plates contained tryptic soy agar or Mueller-Hinton (MH) agar (Becton Dickinson and Co., Sparks, MD).

Selective media plates (MH agar) contained 100 g/mL sodium azide and one of the following antibiotics: ciprofloxacin (0.125 g/mL); sulfamethoxazole (100 g/mL); or chloramphenicol (40 µg/mL).

Results and discussion

All 21 isolates were resistant to nalidixic acid [minimum inhibitory concentration (MIC)  32 g/mL)] and ciprofloxacin (MIC  4 g/mL). Nineteen isolates were intermediate resistant (8 g/mL) and two were resistant (16 g/mL) to norfloxacin. Six strains were identified as being ESBL-producers.

A c c e p t e d

M a n u s c r i p t 7 Fifteen of the twenty-one isolates tested positive for the presence of qnr genes (Table 1). Twelve carried qnrA1, which in eight isolates was found downstream of In37 and ISCR1 that codes for a transposase and provides the promoter sequences for high-level expression of the plasmid-mediated qnrA gene in Enterobacteriaceae. All isolates co-harbouring qnrA, aac(6')-Ib-cr and bla OXA-30 also harboured ISCR1 and a class 1 integron. Three isolates carried qnrB alone. The qnrS gene was not detected. Previous reports describe the expression of qnrA and qnrS [START_REF] Cavaco | First detection of plasmid-mediated quinolone resistance (qnrA and qnrS) in Escherichia coli strains isolated from humans in Scandinavia[END_REF], and the co-existence of qnrB4 and qnrS in the same K. pneumoniae isolate in two different plasmids has also been reported [START_REF] Wang | Occurrence of qnr-positive clinical isolates in Klebsiella pneumoniae producing ESBL or AmpC-type -lactamase from five pediatric hospitals in China[END_REF]. However, here we report four isolates positive for both qnrA and qnrB simultaneously (KP4, KP5, KP6 and KP10). Digestion with S1 revealed the presence of plasmids ranging in size from 50 kb to 450 kb in the strains possessing both genes (data not shown). S1-restricted plasmids were hybridised with labelled qnrA-and qnrB-specific probes revealing that both genes are plasmid-encoded and that qnrA1 and qnrB4 are located on the same plasmid in two of the strains (KP4 and KP10) and on different plasmids in two strain (KP5 and KP6) (data not shown). The qnrA1 gene was found on a 450 kb plasmid in strains KP4, KP5, KP6 and KP10. The qnrB4 gene was located on the same 450 kb plasmid in two of the strains (KP4 and KP10) and on a 75 kb plasmid in strains KP5 and KP6. This is the first report of the presence of both qnrA1 and qnrB4 on the same plasmid. Conjugation was attempted using E. coli J53 Azi r as recipient, however none of the qnr genes could be transferred under the different experimental laboratory conditions tested. -Lactamase screening in all isolates revealed that bla TEM was present in 38% of the isolates and bla SHV in 85.7%. bla OXA was found in 38% of the A c c e p t e d M a n u s c r i p t 8 isolates and was found to be part of the In37 array in all of them. Interestingly, although a genetic linkage between bla CTX-M and aac(6')-Ib-cr has been reported previously [START_REF] Machado | Portuguese Resistance Study Group. Dissemination in Portugal of CTX-M-15-, OXA-1-, and TEM-1-producing Enterobacteriaceae strains containing the aac(6')-Ib-cr gene, which encodes an aminoglycoside-and fluoroquinolone-modifying enzyme[END_REF][START_REF] Cordeiro | Ciprofloxacin-resistant enterobacteria harboring the aac(6')-Ib-cr variant isolated from feces of inpatients in an intensive care unit in Uruguay[END_REF][START_REF] Kim | Prevalence and characteristics of aac(6')-Ib-cr in AmpC-producing Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens: a multicenter study from Korea[END_REF], bla CTX-M was not detected. However, all cephalosporin-resistant isolates harboured qnr genes, thus suggesting a linkage between the presence of qnr and resistance to cephalosporins as previously reported [START_REF] Nordmann | Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae[END_REF][START_REF] Cavaco | First detection of plasmid-mediated quinolone resistance (qnrA and qnrS) in Escherichia coli strains isolated from humans in Scandinavia[END_REF]. Strains KP4 and KP5, although negative for bla CTX-M , harboured qnrA, qnrB and bla DHA-1 encoding a plasmidic AmpC-type lactamase that confers resistance to cephamycins and extended-spectrum cephalosporins, explaining the resistance to cephalosporins exhibited by these isolates. Moreover, intI1 was detected in 80% of the isolates, and 38% carried more than one integron. A total of 18 gene cassettes occurring in three different cassette arrays were identified, with the variants of aadA and dfrA genes being most frequent. The complex integron In37 aac(6')-Ib-cr, bla OXA- 30 , catB3, arr-3:ISCR1:qnrA1 was found in eight isolates; this structure has been previously described in an E. coli isolate from China (accession no. AY259086). Our results showed a high association between aac(6')-Ib-cr, bla OXA-30 , class 1 integron, ISCR1 and qnrA, as previously reported [START_REF] Kim | Prevalence and characteristics of aac(6')-Ib-cr in AmpC-producing Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens: a multicenter study from Korea[END_REF], suggesting the dissemination of this structure worldwide. Also, most of the qnr-positive isolates carried a bla gene encoding an ESBL or an ampC gene coding DHA-1, reinforcing once again a possible relationship between qnr genes and broad spectrum -lactamases [START_REF] Nordmann | Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae[END_REF][START_REF] Pai | Association of qnrB determinants and production of extended-spectrum -lactamases or plasmid-mediated AmpC -lactamases in clinical isolates of Klebsiella pneumoniae[END_REF]. These findings indicate an extremely high prevalence of qnr genes associated with various mobile elements such as ISCR1 and class 1 integrons. Moreover, the role of coexistence of both qnrA and qnrB for the strain still needs to be clarified,

  study, 21 multidrug-resistant Klebsiella pneumoniae isolates were recovered from patients hospitalised in the Intensive Care Unit of Hospital Infante D. Pedro in Aveiro, Portugal. Fifteen isolates carried qnr genes. Four strains harboured the quinolone resistance genes qnrA and qnrB, both located on a large plasmid in two strains (KP4 and KP10) and on different plasmids in two strains (KP5 and KP6). These findings indicate an extremely high prevalence of qnr genes associated with various mobile elements such as ISCR1 and class 1 integrons.

  from patients in the Intensive Care Unit of Hospital Infante D.

  Chromosomal and plasmid DNA were separated by electrophoresis in a 1% agarose gel in 0.5  TBE buffer (pH 8.0) at 200 V and 14 C for 20 h, with the pulse time ramped from 5 s to 45 s. The resulting pulsed-field gel electrophoresis (PFGE) gel was used to assess the qnr gene location, which was determined by DNA-DNA hybridisation using a radiolabelled probe consisting of polymerase chain reaction (PCR)-generated qnrA and qnrB genes.

  A c c e p t e d M a n u s c r i p t 6 2.4. Polymerase chain reaction and DNA sequencing

A

  c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

Acknowledgment

The authors thank the Department of Medical Microbiology of Hospital Infante D. Pedro EPE (Aveiro, Portugal) for the strains.

Funding SF was funded by a fellowship (SFRH/BDE/15573/2005) from Fundação para a Ciência e a Tecnologia de Portugal and by Hospital Infante D. Pedro. EPE (Aveiro, Portugal).

A c c e p t e d M a n u s c r i p t 9 however it can act as a reservoir that will further disseminate these genes to sensitive bacteria.

Competing interests

None declared. Characteristics of qnrA-, qnrB-and qnrA/qnrB-positive Klebsiella pneumoniae strains Isolate MIC (g/mL) Genetic element detected

Ethical approval

Not required

MIC, minimum inhibitory concentration; NA, nalidixic acid; CIP, ciprofloxacin; NOR, norfloxacin; -, negative for the presence of the screened gene; +, positive for the presence of the screened gene.