Mickaël Riou

Sylviane Carbonnelle

Laëtitia Avrain

Narcisa Mesaros

Jean-Paul Pirnay

Florence Bilocq

Daniel De Vos

Anne Simon

Denis Piérard

Frédérique Jacobs

Anne Dediste

Franc ¸oise Paul M Tulkens
email: tulkens@facm.ucl.ac.be

Van Bambeke

Youri Glupczynski

Françoise Van Bambeke

In vivo development of antimicrobial resistance in Pseudomonas aeruginosa strains isolated from the lower respiratory tract of Intensive Care Unit patients with nosocomial pneumonia and receiving antipseudomonal therapy

Keywords:

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In vivo development of antimicrobial resistance in

Pseudomonas aeruginosa strains isolated from the lower respiratory tract of Intensive Care Unit patients with nosocomial pneumonia and receiving antipseudomonal therapy

Care Unit (ICU) patients, with an increased prevalence of multiresistant strains. We examined the impact of the use of antipseudomonal antibiotic(s) on the susceptibility of P. aeruginosa isolated from ICU patients with clinically suspected hospital-acquired pneumonia collected in five teaching hospitals (110 non-duplicate initial isolates; 62 clonal pairs of initial and last isolates during treatment). Minimum inhibitory concentrations (MICs) were determined for amikacin, ciprofloxacin, meropenem, piperacillin/tazobactam (TZP), cefepime and ceftazidime (used in therapy) as well as five reporter antibiotics (aztreonam, colistin, gentamicin, piperacillin and ticarcillin) using Clinical and Laboratory Standards Institute (CLSI) methodology. Susceptibility was assessed according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) and CLSI breakpoints. Resistance rates prior to treatment exceeded 25% for cefepime, ceftazidime, piperacillin, ticarcillin and aztreonam (EUCAST and CLSI) and for gentamicin, TZP and colistin (EUCAST only).

The highest rates of cross-resistance were noted for ceftazidime and cefepime and the lowest rate for amikacin. Mean MIC values were systematically higher in isolates from patients previously exposed (1 month) to the corresponding antibiotic. For clonal pairs, a systematic increase in MIC between initial and last isolates (significant for amikacin, cefepime, meropenem and TZP) was noted. There was a significant correlation between the use of antibiotics (adjusted for respective proportional use of each drug) and loss of susceptibility at the population level when using EUCAST breakpoints. The high level of resistance of P. aeruginosa in ICU patients with A c c e p t e d M a n u s c r i p t

Introduction

Pseudomonas aeruginosa is a major nosocomial pathogen [START_REF] Richards | Nosocomial infections in medical intensive care units in the United States. National Nosocomial Infections Surveillance System[END_REF]. One of its preferential niches is the respiratory tract of Intensive Care Unit (ICU) patients with severe co-morbidities and receiving antibiotic treatment(s), resulting in so-called hospital-acquired pneumonia (HAP), especially in patients with impaired host defences [START_REF] Agodi | Pseudomonas aeruginosa carriage, colonization, and infection in ICU patients[END_REF][START_REF] Restrepo | The role of Gram-negative bacteria in healthcareassociated pneumonia[END_REF][START_REF] Poch | What are the important risk factors for healthcareassociated pneumonia?[END_REF][START_REF] Garau | Pseudomonas aeruginosa pneumonia[END_REF]. The need for early appropriate antibiotic treatment in these patients [START_REF] Bodmann | Current guidelines for the treatment of severe pneumonia and sepsis[END_REF] is substantiated by the observation of a direct correlation between increase in mortality rates and the delay with which such treatment is initiated [START_REF] Rello | Pneumonia in the intensive care unit[END_REF][START_REF] Sandiumenge | Therapy of ventilator-associated pneumonia. A patient-based approach based on the ten rules of 'The Tarragona Strategy[END_REF][START_REF] Kollef | Predictors of 30-day mortality and hospital costs in patients with ventilatorassociated pneumonia attributed to potentially antibiotic-resistant Gramnegative bacteria[END_REF]. Pseudomonas aeruginosa has a remarkable ability to develop resistance to most antimicrobial agents through multiple mechanisms. In this context, the last decades have witnessed the rapid and worldwide emergence of multidrug resistance in P. aeruginosa, with strains developing acquired resistance to almost all available classes of antipseudomonal antibiotics, including broad-spectrum penicillins, cephalosporins, carbapenems, aminoglycosides and fluoroquinolones (see [START_REF] Quinn | Emergence of resistance to imipenem during therapy for Pseudomonas aeruginosa infections[END_REF] for a first historical report regarding imipenem, [START_REF] Fish | Development of resistance during antimicrobial therapy: a review of antibiotic classes and patient characteristics in 173 studies[END_REF] for a review of 173 studies until the early 1990s and [START_REF] Kollef | Predictors of 30-day mortality and hospital costs in patients with ventilatorassociated pneumonia attributed to potentially antibiotic-resistant Gramnegative bacteria[END_REF][START_REF] Carmeli | Emergence of antibiotic-resistant Pseudomonas aeruginosa: comparison of risks associated with different antipseudomonal agents[END_REF][START_REF] Zhuo | Increased mortality of ventilated patients with endotracheal Pseudomonas aeruginosa without clinical signs of infection[END_REF][START_REF] Vettoretti | Emergence of extensive-drug-resistant Pseudomonas aeruginosa in a French university hospital[END_REF] for selected more recent reports).

Acquisition of resistance is multifactorial, with mechanisms as varied as changes in membrane permeability, active efflux, production of antibioticdegrading enzymes, and target mutations [START_REF] Vettoretti | Emergence of extensive-drug-resistant Pseudomonas aeruginosa in a French university hospital[END_REF][START_REF] Pirnay | Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolates[END_REF][START_REF] Mesaros | Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium[END_REF][START_REF] Nakamura | The roles of the quorum-sensing system in the release of extracellular DNA, lipopolysaccharide, and membrane vesicles from Pseudomonas aeruginosa[END_REF]. Infections with resistant strains are a major concern because they increase the risk of therapeutic failure [START_REF] Sabuda | Utilization of colistin for treatment of multidrug-resistant Pseudomonas aeruginosa[END_REF][START_REF] Maeda | Antimicrobial effects of drugs against multidrug-resistant Pseudomonas aeruginosa[END_REF] and are associated with secondary bacteraemia [START_REF] Ammerlaan | Quantifying changes in incidences of nosocomial bacteraemia caused by antibiotic-susceptible and antibiotic-resistant pathogens[END_REF] and a considerable increase in mortality, length of hospital stay and overall health costs [START_REF] Kollef | Predictors of 30-day mortality and hospital costs in patients with ventilatorassociated pneumonia attributed to potentially antibiotic-resistant Gramnegative bacteria[END_REF][START_REF] Giamarellou | Prescribing guidelines for severe Pseudomonas infections[END_REF][START_REF] Sostarich | Impact of multiresistance of Gram-negative bacteria in bloodstream infection on mortality rates and length of stay[END_REF].

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

The emergence of multidrug-resistant (MDR) phenotypes makes epidemiological surveillance of resistance increasingly essential for the appropriate choice of empirical antibiotic regimens. Longitudinal surveillance may be even more important since there is increasing evidence that P. aeruginosa is capable of developing resistance to antibiotics during treatment [START_REF] Carmeli | Emergence of antibiotic-resistant Pseudomonas aeruginosa: comparison of risks associated with different antipseudomonal agents[END_REF][START_REF] Cometta | Prospective randomized comparison of imipenem monotherapy with imipenem plus netilmicin for treatment of severe infections in nonneutropenic patients[END_REF][START_REF] Jaccard | Prospective randomized comparison of imipenem-cilastatin and piperacillin-tazobactam in nosocomial pneumonia or peritonitis[END_REF][START_REF] Harris | Risk factors for piperacillin-tazobactam-resistant Pseudomonas aeruginosa among hospitalized patients[END_REF].

In the present study, the level of in vitro resistance of P. aeruginosa isolates obtained at the onset of therapy and during treatment (clonal pairs) from patients with clinically suspected nosocomial pneumonia for which microbiological cultures strongly suggested that P. aeruginosa was the causative organism and who, accordingly, were treated with antipseudomonal antibiotics was assessed. A high rate of initial resistance to all antibiotics used in this set-up was observed, except for amikacin, as well as an increase in resistance of the same clonal isolates during treatment in relation to the global use of antibiotics in this population.

Materials and methods

Overall study design, patient selection, clinical analysis, record of antibiotic prescription and use, and time frame

The protocol of this observational study (no deviation from the standard of care of patients), as approved by the Ethical Committee of the Faculty of Medicine, Université Catholique de Louvain (Brussels, Belgium), was to enrol prospectively patients with a clinical diagnosis of nosocomial pneumonia

Isolates used for the study, reference strains, minimum inhibitory concentration (MIC) determination and susceptibility criteria

Each isolate received by the co-ordinating laboratory was checked for purity and for the presence of a single clone based on colony morphology. When needed, identification was checked using a commercial gallery (API ® 20 NE; bioMérieux, Marcy l'Etoile, France) and by ability to grow at 42 C.

Pseudomonas aeruginosa reference strains ATCC 27853 and PAO1 [START_REF] Stover | Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen[END_REF] were used as internal quality controls. MICs were determined by geometric microdilution in cation-adjusted Mueller-Hinton broth (BD Diagnostics, Franklin Lakes, NJ) according to Clinical and Laboratory Standards Institute (CLSI) recommendations [START_REF]Performance standards for antimicrobial susceptibility testing[END_REF]. Susceptibility categorisation was assessed A c c e p t e d M a n u s c r i p t according to current susceptibility and resistance breakpoints of the European Committee on Antibiotic Susceptibility Testing (EUCAST) [START_REF]Breakpoint tables for interpretation of MICs and zone diameters: version 1[END_REF] and the CSLI [START_REF]Performance standards for antimicrobial susceptibility testing. Twentieth informational supplement[END_REF]. A threshold criterion of 95% similarity was used, corresponding to two or less peak differences in the whole electrophoresis pattern. The same method was used to assess the clustering of initial (D0) samples in either the much conserved clonal serotype O12 or in one of four serotype O11 clonal complexes (CCs) (F, G, H and I).

Determination of clonality

Materials for laboratory studies

Gentamicin, amikacin, ticarcillin, piperacillin, ciprofloxacin and colistin (polymyxin E) were obtained from Sigma-Aldrich (St Louis, MO); aztreonam and cefepime (Bristol-Myers Squibb, Brussels, Belgium), meropenem (AstraZeneca, Brussels, Belgium), ceftazidime (GlaxoSmithKline, Genval, Belgium) and piperacillin/tazobactam (TZP) (Wyeth Pharmaceuticals, Ottignies-Louvain-la-Neuve, Belgium) were obtained as the corresponding branded products registered for intravenous administration and complying with the provisions of purity and content of the European Pharmacopoeia. All other chemicals were of analytical grade and were obtained from E. Merck AG (Darmstadt, Germany) or Sigma-Aldrich. All culture media were from BD Diagnostics.

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

Statistical methods

Statistical analyses were performed using GraphPad ® Prism software v. 4.3 and GraphPad InStat v.3.06 (GraphPad Software Inc., La Jolla, CA) and using the online statistical calculator from the Saint John's University (Collegeville, MI) [START_REF] Kirkman | Statistics to use[END_REF] for testing the normality of the MIC distributions included in each comparison [two-sample Kolmogorov-Smirnov test of normality, with calculation of cumulative probabilities (KS P)].

Results

Overall study design, characteristics of samples and patients, general clinical data and treatments

Fig. 1 shows the origin and mode of selection of the isolates analysed in this study. From an initial 144 patients [identified as being hospitalised in the ICUs of the participating hospitals and from whom a P. aeruginosa strain had been isolated (233 non-duplicate isolates)], 104 patients (199 non-duplicate isolates) were retained as fulfilling the clinical and radiological criteria for suspicion of nosocomial pneumonia and with P. aeruginosa likely to be the main aetiological agent. From these 104 patients, 110 non-duplicate initial isolates were obtained (referred to as D0 samples). To exclude biases due the potential presence of epidemic multiresistant clones, all isolates were genotyped by the semi-automated REP-PCR-based DiversiLab method.

Whether isolates clustered in the very conserved serotype O12 clone or in one of four serotype O11 CCs (F, G, H and I), known to be the most frequently involved in outbreaks caused by MDR P. aeruginosa strains, was A c c e p t e d M a n u s c r i p t also analysed [START_REF] Pirnay | Pseudomonas aeruginosa population structure revisited[END_REF]. Only two isolates could be associated with the epidemic MDR O12 clone, but they were isolated in different hospitals at a 2-year interval. Six isolates could be associated with one of the O11 CCs, but again these were from different hospitals and obtained at several months interval.

For 69 patients, successive isolates were obtained during antipseudomonal treatment, resulting in 62 confirmed clonal pairs of an initial and a last isolate (the latter being referred to as DL samples). Patients were mostly adults (only three patients were <18 years of age and excluding them did not change the results of the analyses) and were very often ventilated at the time of the diagnosis (Table 1). For patients from whom clonal pairs could be obtained: (i) approximately one-half received monotherapy only; (ii) in general, amikacin (often for a short period only and always in combination with another antibiotic), meropenem, cefepime and TZP were preferentially prescribed; and

(iii) approximately one-third of the patients died, but only one-half of them from the infection. It was also observed that in all patients, initial antibiotic dosages and schedules were (i) those recommended for severe infections and (ii) according to their pharmacokinetic/pharmacodynamic properties (see details in Table 1). Analysis of the clinical records performed independently of the attending clinicians showed no systematic bias in the way follow-up samples were obtained between patients and centres. for meropenem and ciprofloxacin and was only 8% for amikacin. More than 25% of the isolates were resistant by EUCAST criteria to all other antibiotics tested for epidemiological purposes. Of note, MICs of colistin were all in a narrow range (1-4 mg/L), i.e. at the limit of the EUCAST breakpoints. There was a high level of cross-resistance between TZP on the one hand and ceftazidime and cefepime on the other hand (in ca. 75% of the TZP-resistant isolates; see Supplementary Table 1). Isolates resistant to colistin according to EUCAST were also often resistant to ciprofloxacin, meropenem, cefepime and ceftazidime (33-42% of colistin-resistant isolates). Fig. 2 shows the impact of previous exposure (up to 1 month) to five antibiotics on the MIC of the initial P. aeruginosa isolates (ceftazidime was excluded because of the small number of patients). For all antibiotics except amikacin, MIC values were systematically higher when patients had been previously exposed to the corresponding antibiotic (with geometric means approaching or even exceeding the EUCAST susceptibility breakpoint).

Susceptibilities of initial isolates

However, this decrease in susceptibility was statistically significant for meropenem and TZP only.

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

Changes in susceptibilities during exposure to antipseudomonal antibiotics and clinical outcomes

Fig. 3 shows the change in susceptibility of clonal isolates between the initial (D0 isolate) and last day (DL isolate) of treatment. The MIC of all antibiotics increased, with the differences reaching statistical significance for all antibiotics (for ciprofloxacin, by considering log 2 transformed data only, probably due to the low number of samples). When assessing each clone individually, it was found that MIC values of most antibiotics increased by twoto four-fold compared with the initial value (1-2 log 2 dilutions) (Fig. 4).

Excluding patients with <5 days of antipseudomonal treatment (8 of 59 patients) did not modify the results. A retrospective case-control study was performed to identify whether an MIC increase could be correlated with administration of the respective antibiotic. Whilst each antibiotic treatment was associated with an odds ratio >1 for MIC increase, this was statistically significant for amikacin only. An attempt to link the decrease in susceptibility to the duration of exposure to any specific antibiotic, or to all of them, did not yield significant results because of the low number of patients in each subgroup.

Table 3 shows that the decreased susceptibility observed during treatment caused marked increases in the proportion of isolates categorised as intermediate or resistant using EUCAST breakpoints, with all of them except amikacin exceeding a threshold of 25%. There was a significant correlation between the proportional use of each antibiotic and the loss of susceptibility at the whole population level. An apparent loss of susceptibility for colistin A c c e p t e d M a n u s c r i p t (based on the EUCAST breakpoint for resistance of >4 mg/L) was also documented. Cross-resistance was also increased (not shown), but this did not reach statistical significance because of the too small number of isolates.

Patients who died from the pneumonia (n = 9) (see Table 1) had not been more exposed to inappropriate antipseudomonal antibiotics during treatment [in terms of proportion of active antibiotics received (12 of 15)] than the general population.

Discussion

The present study represents one of the first recent efforts to document systematically the loss of susceptibility of P. aeruginosa isolates to antipseudomonal antibiotics when used in the treatment of clinically suspected HAP for which P. aeruginosa was considered the putative causative organism.

Making a diagnosis of pneumonia in the ICU is notoriously difficult [START_REF] Andrews | Diagnosis of nosocomial bacterial pneumonia in acute, diffuse lung injury[END_REF] since radiographic signs of chest infiltrates as well as microbiological analysis both lack specificity. Furthermore, collection of deep invasive specimens by bronchoscopy is often not feasible in these mechanically ventilated patients because of their unstable condition. Because this study was observational, it was not possible to obtain true quantitative cultures from all patients as this would have exceeded the current standard of care. Thus, we are left with some degree of uncertainty about the true pseudomonal nature of the infection in some episodes. However, since all enrolled patients were treated with antipseudomonal antibiotics, the main goal of our study, which was to examine the increase in resistance of P. aeruginosa in patients (i) from the A c c e p t e d M a n u s c r i p t onset of their antipseudomonal treatment and (ii) for whom clonal pairs could be isolated during this treatment was actually reached, irrespective of whether the true causative organism was P. aeruginosa. Potential biases due to the presence of known multiresistant epidemic clones were explicitly excluded, analysing in detail the MIC shifts occurring for clonal pairs obtained during exposure to antipseudomonal antibiotics and applying the interpretative criteria of EUCAST. Access to follow-up samples was limited by the decision of the clinician as to whether to perform a second or more subsequent samplings during therapy owing to ethical and practical considerations. Whilst this may have led to lack of samples from patients with a rapid fatal outcome (which could have heralded gross antibiotic failure), the converse may also be true, i.e. patients with rapid improvement, perhaps due to antibiotic(s), are less likely to yield more than an initial sample. The data show that (i) empirical therapy, assuming that P. aeruginosa is the causative organism, is often inappropriate in terms of choice of antibiotic (with pre-exposure to the same antibiotic being a detrimental element, except for amikacin) and (ii) increase in resistance occurs for all antibiotics during exposure.

Treatment of pneumonia caused by P. aeruginosa is difficult, with crude mortality rates reaching 40% or higher [START_REF] Chastre | Ventilator-associated pneumonia[END_REF]. In the present series, the mortality rate of patients for whom successive clonal samples could be obtained reached ca. 30%, with inability to control the infection being the main likely cause of death for approximately one-half of these patients. In contrast to an A c c e p t e d M a n u s c r i p t earlier report in which rates of primary resistance of P. aeruginosa in the ICU were relatively low [START_REF] Juan | Contribution of clonal dissemination and selection of mutants during therapy to Pseudomonas aeruginosa antimicrobial resistance in an intensive care unit setting[END_REF], here it was found, as in another recent study [START_REF] Solh | Update on the treatment of Pseudomonas aeruginosa pneumonia[END_REF], that MDR organisms are frequent at the very early onset of the disease. Although not designed to provide a true epidemiological estimate, the present study clearly shows that the clinician's choice of active antibiotics has become increasingly narrow when P. aeruginosa is among the causative organisms.

Thus, combined empirical therapy, although still a matter of debate [START_REF] Chastre | Comparison of 8 vs 15 days of antibiotic therapy for ventilator-associated pneumonia in adults: a randomized trial[END_REF][START_REF] Bassetti | Pseudomonas aeruginosa serious infections: mono or combination antimicrobial therapy?[END_REF][START_REF] Paul |  Lactam monotherapy versus  lactam-aminoglycoside combination therapy for sepsis in immunocompetent patients: systematic review and meta-analysis of randomised trials[END_REF], may now be the only available option to ensure a reasonable coverage if P. aeruginosa is considered to be the aetiological agent. As was the case in this study, combination therapy is actually often used in daily practice and is advocated as being essential to obtain a satisfactory response [START_REF] Solh | Update on the treatment of Pseudomonas aeruginosa pneumonia[END_REF][START_REF] Lode | Lower respiratory tract infections in the intensive care unit: consequences of antibiotic resistance for choice of antibiotic[END_REF]. A first main conclusion from this study is, therefore, that significant efforts must be deployed to accelerate the early assessment of bacterial susceptibility in order to decrease the risk of therapeutic failure [START_REF] Parker | Ventilator-associated pneumonia caused by multidrug-resistant organisms or Pseudomonas aeruginosa: prevalence, incidence, risk factors, and outcomes[END_REF] while at the same time avoiding unnecessary use of wide-spectrum combinations.

As anticipated from previous recent studies [START_REF] Juan | Contribution of clonal dissemination and selection of mutants during therapy to Pseudomonas aeruginosa antimicrobial resistance in an intensive care unit setting[END_REF][START_REF] Solh | Update on the treatment of Pseudomonas aeruginosa pneumonia[END_REF][START_REF] Depuydt | Determinants and impact of multidrug antibiotic resistance in pathogens causing ventilator-associated-pneumonia[END_REF], a clear trend towards an increase in resistance of the initial isolates during treatment was also observed. In addition, results from the present study demonstrate that therapeutic choices if P. aeruginosa is among the target organisms are narrowed down considerably when EUCAST interpretative criteria are endorsed. Although the decreased susceptibilities observed in the present study were often not statistically significant, this should not undermine the conclusions. The antibiotic doses and treatment schedules used (all at or close to the maximum values set forth in the respective labellings; see A c c e p t e d M a n u s c r i p t footnote a in Table 1) as well as the frequent use of combinations were actually expected to decrease the risk of emergence of resistance and/or selection of less susceptible subpopulations [START_REF] Mesaros | Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium[END_REF][START_REF] Craig | Basic pharmacodynamics of antibacterials with clinical applications to the use of -lactams, glycopeptides, and linezolid[END_REF][START_REF] Craig | Once-daily versus multiple-daily dosing of aminoglycosides[END_REF][START_REF] Van Bambeke | Quinolones in 2005: an update[END_REF][START_REF] Gerber | Selection of aminoglycoside-resistant variants of Pseudomonas aeruginosa in an in vivo model[END_REF][START_REF] Kiem | Impact of organism species on microbial eradication and development of resistance in severe Gram-negative pneumonia[END_REF][START_REF] Kiem | Relationship of minimal inhibitory concentration and bactericidal activity to efficacy of antibiotics for treatment of ventilatorassociated pneumonia[END_REF]. For obvious ethical reasons, a study in which a significant proportion of patients would be treated in a suboptimal fashion is, nowadays, impossible to design in a prospective way given what we know about optimisation of antibiotic use.

Thus, the trends we see may actually be the only, but important, signals heralding the risks associated with antibiotic therapy of pseudomonal infection. Of note, short-course amikacin therapy (to minimise the risk of nephrotoxicity) may have contributed to the maintenance of its overall activity towards initial isolates.

Because P. aeruginosa isolates collected from hospitalised patients may originate from multiple sources, it is often difficult to distinguish between emergence of resistance within the original population from the acquisition of another less susceptible strain. Clonal analysis of successive isolates of P. aeruginosa was previously used to address this issue [START_REF] Juan | Contribution of clonal dissemination and selection of mutants during therapy to Pseudomonas aeruginosa antimicrobial resistance in an intensive care unit setting[END_REF][START_REF] Lambiase | Typing of Pseudomonas aeruginosa isolated from patients with VAP in an intensive care unit[END_REF]. The method used here targets highly conserved non-coding repetitive sequences [START_REF] Healy | Microbial DNA typing by automated repetitive-sequence-based PCR[END_REF] and thus ensures a higher level of reliability. This study therefore provides overwhelming evidence that the decrease in susceptibility of P. aeruginosa observed in patients receiving antipseudomonal antibiotics may really take place within the original bacterial population. Although the precise mechanisms that cause these changes in susceptibility still need to be studied in detail, the moderate increases in MIC would suggest a predominant role of increased efflux or decreased porin permeability [START_REF] Hancock | Function of Pseudomonas porins in uptake and efflux[END_REF][START_REF] Van Bambeke | Antibiotic efflux pumps in prokaryotic cells: occurrence, impact on resistance and strategies for the future of antimicrobial therapy[END_REF]. A second main A c c e p t e d M a n u s c r i p t conclusion of this study is therefore that close monitoring of susceptibility testing should be performed during treatment since even minor changes may result in a change of susceptibility categorisation when using EUCAST breakpoints. Based on the most likely underlying antibiotic resistance mechanism, they also may lead to cross-resistance between structurally very different antibiotic classes [START_REF] Van Bambeke | Antibiotic efflux pumps in prokaryotic cells: occurrence, impact on resistance and strategies for the future of antimicrobial therapy[END_REF][START_REF] Poole | Efflux-mediated multiresistance in Gram-negative bacteria[END_REF][START_REF] Lister | Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms[END_REF]. Both considerations should lead to important reassessment of the therapeutic strategies. In this context, the present data on colistin are interesting as they rationalise the recent EUCAST breakpoint (resistant >4 mg/L) adaptation. At Day 0 we were mainly confronted with an essentially wild-type population (few patients if any had received colistin), and yet the former EUCAST breakpoint (resistant >2 mg/L) would have categorised almost one-half of this population as resistant. The rise in colistin MIC observed in DL isolates is a reason for concern as this drug was almost never used for treatment. More broadly speaking, these results, and those of many other studies, clearly call for the design and use of new molecules with a lesser propensity to trigger the emergence of resistance.

Throughout this study we were faced with the difficulty of choosing appropriate criteria, namely those of EUCAST or CLSI, for categorising isolates as susceptible, intermediate or resistant to the antibiotics under study.

In vitro criteria of this kind are only useful as long as they provide reasonably accurate predictive information about the clinical outcome of therapy.

However, this study was not designed to validate their accuracy for prediction of clinical outcomes. Further studies focusing on specific antibiotics will be A c c e p t e d M a n u s c r i p t needed to allow for a rational and final choice between these two common sets of interpretative criteria.

In summary, this study demonstrates that resistance of P. aeruginosa to commonly recommended antipseudomonal antibiotics is an every-day reality in the present environment of ICUs and that current standard therapies do not prevent an increase in resistance during exposure to these antibiotics. Whilst only P. aeruginosa was studied here, a similar situation may prevail for other Gram-negative bacteria causing nosocomial pneumonia, such as Klebsiella spp., Enterobacter spp. or Acinetobacter baumannii. These are indeed emerging rapidly, in part as a consequence of prior antibiotic therapy [START_REF] Rello | Microbial causes of ventilator-associated pneumonia[END_REF], and share the capacity of becoming resistant to many first-line antibiotics.

Observational studies coupled to the development of early diagnostic methods therefore seem warranted. For the statistical analysis, the distributions were subjected to normality test [Kolmogorov-Smirnov (KS)] and were found to be unlikely to be normally distributed when using the raw data, but normally distributed for ciprofloxacin (KS P > 0.59), consistent with a normal distribution for meropenem, TZP and cefepime (KS P = 0.23-0.41) and unlikely to be normally distributed for amikacin [KS P (at D0) = 0.05) when using their log 2 transformed data. The differences in MICs between each of the two sets of samples (D0 vs. DL) was therefore examined both by a parametric (two-tailed paired t-test) and a non-parametric (Wilcoxon matched-pair test) using both raw data and their log 2 transforms. Differences were found to be significant both for raw and log 2 transformed data by both tests (marked as *) for meropenem (P = 0.011 and 0.002) and TZP (P = 0.028 and 0.008) and by Wilcoxon test only (marked as a) for amikacin (P = 0.013) and cefepime (P = 0.009). For ciprofloxacin, the difference was not statistically significant (P > 0.05 for both tests) when considering raw data, but was significant (P < 0.02 for both tests) when using their log 2 transforms (marked as [*]). c From the CLSI website (http://www.clsi.org) [START_REF]Performance standards for antimicrobial susceptibility testing. Nineteenth informational supplement[END_REF]; breakpoints (clinical;

isolates with MIC >S and <R are considered intermediate).

d Antibiotics commonly used for antipseudomonal treatment.

e Antibiotics used for epidemiological purposes in the context of the present study.

f No intermediate category clinical breakpoints for this antibiotic.

g According to the CLSI, the S category for TZP or PIP relates to high-dose therapy for serious infections and monotherapy is associated with treatment failure in serious infections.

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  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 ABSTRACT Pseudomonas aeruginosa causes severe nosocomial pneumonia in Intensive

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  c c e p t e d M a n u s c r i p t nosocomial pneumonia as well as its further increase during treatment severely narrows the already limited therapeutic options. Further observational studies and the development of early diagnosis for resistant isolates are warranted.

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  c c e p t e d M a n u s c r i p t (defined as not present or incubating at the time of admission to the hospital and occurring >48 h later) based on clinical findings (fever, increase in volume of bronchial secretions, inflammatory syndrome with leukocytosis) along with the appearance of new radiographic infiltrates [after exclusion of other noninfectious causes of chest infiltrates such as alveolar haemorrhage due to trauma or other causes unrelated to infection (such as drug toxicity or acute respiratory distress syndrome)] and showing the presence of P. aeruginosa in endotracheal aspirates, bronchoalveolar lavage or puncture samples such as pleural fluid, empyema or blood cultures. Cultures were quantitative in some centres and semiquantitative in others [i.e. grading of bacterial growth as heavy (+4), moderate (+3), light (+2) or rare (+1) according to the growth density following streaking of the culture plates in four quadrants]. When multiple microorganisms were present, the role of P. aeruginosa as the likely aetiological pathogen was only retained if it appeared as the predominant organism. Cystic fibrosis patients were excluded. A complete retrospective analysis of the clinical charts was made to collect information on prior and current antibiotic regimens (during the pneumonia episode) as well as overall treatment outcome. Since suboptimal therapies are considered to promote the emergence of resistance, the quality of the treatments used was examined in terms of dosages and schedules of administration and was compared with (i) those recommended in the corresponding official Belgian labelling (also known as Summary of Product Characteristics) for severe infections and (ii)those based on accepted pharmacokinetic/pharmacodynamic criteria for optimised therapy foe the corresponding antibiotics[START_REF] Craig | Basic pharmacodynamics of antibacterials with clinical applications to the use of -lactams, glycopeptides, and linezolid[END_REF][START_REF] Craig | Once-daily versus multiple-daily dosing of aminoglycosides[END_REF][START_REF] Van Bambeke | Quinolones in 2005: an update[END_REF].

  Sample collection was performed in five Belgian teaching hospitals (four in the Brussels region and one in Wallonia region) and was initiated in 2006, although most samples were collected during the period 2007-2009. A sample was obtained at the time of initial diagnosis (D0 samples) for all enrolled patients (104 patients; 110 initial isolates). For 69 patients, a second (or more) subsequent sample(s) could be obtained during the course of therapy (range 1-123 days; mean 23 days; median 17.5 days) based on the decision of the clinician to perform such additional sampling as part of their standard of care. Bacterial identification was carried out locally using standard microbiological methods, after which samples were frozen in cryovials at -80 C for transfer to the co-ordinating laboratory (Université Catholique de Louvain).

  Clonality of successive isolates obtained from individual patients was assessed by repetitive extragenic palindromic-polymerase chain reaction (REP-PCR). Pseudomonas aeruginosa isolates were cultured overnight at 37 C on Luria-Bertani agar (Sigma-Aldrich, St Louis, MO) plates. Total bacterial DNA was extracted using an UltraClean TM Microbial DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA) as detailed in the manufacturer's protocols. REP-PCR profiles of the P. aeruginosa isolates were obtained using a DiversiLab TM system (bioMérieux). The PCR mixture (25 L final volume) contained 11.5 L of sterile distilled water, 1.25 L of GeneAmp ® 10 PCR buffer I (Applied Biosystems, Life Technologies, Carlsbad, CA), 9 L of REP-PCR MM1 (bioMérieux), 1 L of Primer Mix (bioMérieux), 0.25 L (1.25 U) of AmpliTaq ® DNA polymerase (Applied Biosystems) and 2 L of template DNA. Thermal cycles included an initial denaturation at 94 C for 2 min, followed by 35 cycles of denaturation at 94 C for 30 s, annealing at 50 C for 30 s and extension at 70 C for 90 s, and a final extension at 70 C for 3 min. REP-PCR profiles were obtained using microfluidic DNA chips (DiversiLab TM LabChip ® ; bioMérieux) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) according to the manufacturers' instructions. REP-PCR fingerprinting profiles were compared using the Web-based A c c e p t e d M a n u s c r i p t DiversiLab software v.3.3.4 (bioMérieux), which uses the Pearson correlation coefficient and the outweighed pair group method.

  A c c e p t e d M a n u s c r i p t first sample (D0), with susceptibility categorisation according to EUCAST and CLSI criteria (see Supplementary Fig.1for a graphical representation of the cumulative MIC distribution). With respect to the main antibiotics used for therapy in the institutions surveyed, resistance (based on EUCAST breakpoints) exceeded 25% for TZP, cefepime and ceftazidime, was ca. 20%

Fig. 1 .

 1 Fig. 1. Flow diagram identifying the origin of the isolates used in the present

Fig. 2 .

 2 Fig. 2. Minimum inhibitory concentrations (MICs) of five antibiotics used in

A

  c c e p t e d M a n u s c r i p t were subjected to normality test [Kolmogorov-Smirnov (KS)] and were found to be unlikely to be normally distributed when using the raw data, but normally distributed for meropenem [KS P (yes group only) = 0.65] and possibly normally distributed for TZP [KS P (yes group only) = 0.22)] when using their log 2 transformed data. The differences in MICs between each of the two sets of samples (no vs. yes) were therefore examined both by a parametric test [unpaired t-test (two-tailed) with Welch's correction] and a non-parametric test (Mann-Whitney) and were found to be: (i) significant for meropenem by Mann-Whitney (P = 0.0009) when considering raw data and by both tests (P = 0.008 and 0.0009, respectively) when considering their log 2 transforms (marked as *); (ii) close to significance (P = 0.0579 by Mann-Whitney both for raw and log 2 transformed distributions) for TZP (marked as [a]), but not for the other antibiotics (P > 0.06 for both tests and both for raw and log 2 transformed data).

Fig. 3 .

 3 Fig. 3. Changes in the minimum inhibitory concentrations (MICs) of five

A

  c c e p t e d M a n u s c r i p t ≤R indicates intermediate; there are no intermediate categories for piperacillin/tazobactam (TZP) and cefepime].

Fig. 4 .

 4 Fig. 4. Increases in the minimum inhibitory concentrations (MICs) of five

A

  c c e p t e d M a n u s c r i p t for MEM, n = 37 for TZP and n = 30 for FEP). AMK, amikacin; CIP, ciprofloxacin; MEM., meropenem; TZP, piperacillin/tazobactam; FEP, cefepime. 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 td

  Assessed after 90 days following the collection date of the first isolate, except for two patients (alive) for whom the observation period was extended to 202 days and 213 days, respectively.

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 tb

  Figures in bold indicatesituations in which resistance to a given antibiotic exceeds 25% of isolates based on the corresponding criteria (EUCAST or CLSI).

A c c e p t e d M a n u s c r i p t 1

 1

Table 2

 2 shows the susceptibility patterns and MIC 50/90 values (MICs for 50% and 90% of the organisms, respectively) of the 110 isolates obtained from the

Table 1

 1 General characteristics of patients, treatments and outcomes Figures indicate the percentage of patients with an isolate non-resistant tothe drug prescribed in the case of monotherapy, or to one, two, three or four of the antibiotics prescribed in case of multiple drug therapy.

	Edited Table 1
	Total population (N = 104)
		Min.	GM	Mean ±	Median Max.
				S.D.
	Age (years)	1.2	54.1	60.0 ±	63.1	85.0
				19.3
	Ventilated (No. of patients)
	Yes	74
	No	30
	Patients with clonal pairs (n = 59)
	Antibiotics with antipseudomonal potential (initial treatment a ) (no. of
	patients)
	AMK	29
	CIP	11
	MEM	28
	TZP	31
	FEP	29
	CAZ	4
	Assessment of adequateness of initial therapy b
		No. of	No. of adequate	% (no.) of patients with
		patients	antibiotics/total	adequate therapy c based on
				breakpoints of:
				EUCAST	CLSI
	Monotherapy 26	1/1	57.7 (15)	73.1 (19)
	2 antibiotics	14	2/2	71.4 (10)	85.7 (12)
			1/2	28.6 (4)	14.3 (2)
	3 antibiotics	13	3/3	38.5 (5)	46.2 (6)
			2/3	30.8 (4)	30.8 (4)
			1/3	23.1 (3)	23.1 (3)

a Typical initial treatments (for adults): AMK, 15 mg/kg every 24 h; CIP, 200c

Table 2

 2 MIC 50 and MIC 90 values and susceptibility patterns (based on EUCAST and CSLI criteria) of the initial isolates (n = 110) of Pseudomonas aeruginosa from patients with clinically suspected nosocomial pneumonia and enrolled in the

	study
	Antibiotic MIC 50/90	% non-susceptible isolates according to:
		(mg/L)	EUCAST		CLSI
			Breakpoint a	%I/R	Breakpoint c	%I/R
			(S/R>) (mg/L)	b	(S/R) (mg/L)	b
	AMK d	4/16	8/16	9/8	16/64	1/7
	CIP d	0.25/8	0.5/1	7/23	1/4	4/18
	MEM d	1/16	2/8	12/24 4/16	3/24
	TZP d	8/128	16/16	34 f	64 g /128	7/12
	FEP d	8/64	8/8	46 f	8/32	17/30
	CAZ d	4/64	8/8	39 f	8/32	6/33
	GEN e	2/64	4 /4	26 f	4/16	10/15
	PIP e	8/128	16/16	36 f	64 g /128	0/26
	TIC e	64/512	16/16	86 f	64/128	0/39
	ATM e	8/32	1/16	68/30 8/32	20/30
	CST e	2/4	4/4	4.5 f	2/8	26/0
	MIC 50/90 , minimum inhibitory concentrations for 50% and 90% of the
	organisms, respectively; EUCAST, European Committee on Antibiotic
	Susceptibility Testing; CLSI, Clinical and Laboratory Standards Institute; S,
	susceptible; R, resistant; I, intermediate; AMK, amikacin; CIP, ciprofloxacin;
	MEM, meropenem; TZP, piperacillin/tazobactam; FEP, cefepime; CAZ,
	ceftazidime; GEN, gentamicin; PIP, piperacillin; TIC, ticarcillin, ATM,
	aztreonam; CST, colistin.

a From the EUCAST website (http://www.eucast.org); breakpoints (clinical; organisms with MIC >S and ≤R are considered intermediate).

Table 3

 3 Comparative susceptibility of clonal isolates obtained from 59 patients with a clinical diagnosis of nosocomial pneumonia before initiation of treatment (D0) and during antipseudomonal treatment (DL)

	Antibiotic Use	Non-susceptible isolates according to:	Loss of susceptibility (%) during treatment b and correlation with
		(%)	EUCAST (%I/R) a	CLSI (%I/R) a	antibiotic use
			D0	DL	D0	DL	EUCAST	CLSI
	AMK c	22.0	1.6/11.3 11.3/16.1 0.0/11.3 4.8/11.3 14.5	4.8
	CIP c	8.3	4.8/25.8 4.8/35.5 3.2/22.6 6.5/29.0 9.7	9.7
	MEM c	21.2	12.9/22.6 14.5/35.5 1.6/22.6 6.5/35.5 14.5	17.7
	TZP c	23.5	33.9 e	53.2 e	0.0/17.7 0.0/32.3 19.5	14.6
	FEP c	22.0	40.3 e	53.2 e	12.9/27.4 8.1/45.2 14.5	12.9
	CAZ c	3.0	35.5 e	46.8 e	8.1/27.4 8.1/38.7 11.3	11.3
							r = 0.89 f (P = 0.03)	r = 0.27 f (P = 0.66)
	GEN d		21.0 e	29.0 e	8.1/12.9 12.9/16.1 8.0	8.0
	PIP d		35.5 e	54.8 e	0.0/24.2 0.0/33.9 19.4	9.7
	TIC d		87.1 e	91.9 e	0.0/37.1 0.0/62.9 4.8	25.8
	ATM d		71.0/24.2 53.2/43.5 24.2/24.2 21.0/43.5 1.6	16.1
	CST d		4.3 e	37.1 e	24.6/0	24.2/0	32.8	0

Acknowledgments

The authors thank the research nurses of the participating institutions for help in collecting clinical and antibiotic usage data as well as Virginie Mohymont, Charlotte Misson and Jonathan Gesels for dedicated technical help.

Funding

MR and NM were recipients of post-doctoral fellowships of the Région Bruxelloise/Brusselse Gewest (within the context of the programme 'Research in Brussels'). MR was also a Post-doctoral fellow of the Belgian Fonds de la Recherche Scientifique (F.R.S.-FNRS). SC was supported by the Belgian

A c c e p t e d M a n u s c r i p t Fonds de la Recherche Scientifique Médicale (F.R.S.M.) (grant no. 3.597.06).

FVB is Maître de Recherches of F.R.S.-FNRS.

Competing interests

None declared.

Ethical approval

The protocol of this academic, non-commercial, observational study was approved by the Ethical Committee of the Faculty of Medicine of the coordinating institution (Université Catholique de Louvain, Brussels, Belgium) within the context of a grant application to the Belgian Fonds de la Recherche Scientifique Médicale (grant no. 3.597.06). e EUCAST has no intermediate category for these antibiotic/P. aeruginosa combinations.

f Non-parametric correlation (Spearman rank) between the % of use of each antibiotic (% of all antibiotic prescriptions) in the whole population (AMK, 24.0; CIP, 9.6; MEM, 20.2; FEP, 15.4; and CAZ, 3.8) and the increase in % of isolates with change in susceptibility (moving from S to I, I to R, or S to R) for the corresponding antibiotic.


Krisztian ; Banyai

Baranya

Fang-Tzy Wu

Krisztián Bányai

Jason C Huang

Ho-Sheng Wu

Feng-Yee Chang

Jyh-Yuan Yang

Agnes Chao

Hsiung

Yhu-Chering Huang

Jen-Shiou Lin

Hwang

Baoming Jiang

Jon R Gentsch

Diverse origin of P[19] rotaviruses in children with acute diarrhea in Taiwan: detection of 3 novel lineages of the G3, G5, and G9 VP7 genes 4 5 6

Keywords: phylogenetic analysis, VP7, VP4, VP6, NSP4 phylogenetic analysis, VP7, VP4, VP6, NSP4 59 60

Diverse origin of P[19] rotaviruses in children with acute

diarrhea in Taiwan: detection of novel lineages of the G3, G5, and G9 VP7 genes

Introduction 61

Group A rotaviruses are the leading cause of severe dehydrating gastroenteritis in young 62 children worldwide. Globally, rotavirus infections account for an estimated 611,000 deaths 63 each year among children < 5 years of age [Parashar et al., 2006]. Rotavirus particles are RNA. The two outer-layer proteins VP7 and VP4 form the basis of the dual classification 66 system of group A rotavirus into G and P types [START_REF] Estes | Rotaviruses[END_REF]. Of the 24 G and 67 31 P genotypes currently known, at least 11 G types and 12 P types, in at least 70 different 68 antigen combinations have been identified in humans [Matthijnssens et al., 2009]. Of these, 5 69 are globally common (G1P [START_REF]strains detected exclusively on the eastern coast of the USA represents 253 the first documented evidence for human infection with G9 strains and could have caused a 254 minor local outbreak[END_REF], G2P[4], G3P [START_REF]strains detected exclusively on the eastern coast of the USA represents 253 the first documented evidence for human infection with G9 strains and could have caused a 254 minor local outbreak[END_REF], G4P [START_REF]strains detected exclusively on the eastern coast of the USA represents 253 the first documented evidence for human infection with G9 strains and could have caused a 254 minor local outbreak[END_REF], and G9P [START_REF]strains detected exclusively on the eastern coast of the USA represents 253 the first documented evidence for human infection with G9 strains and could have caused a 254 minor local outbreak[END_REF]), while others are 70 unusual combinations of these common antigen types and may be only regionally important. 71

Rare strains include mainly those antigen specificities that originate from periodic occurrence 72 of animal-to-human interspecies transmission events. Examples for such strains include G5, 73 G6, G8, and G11 VP7 genes or P[3], P [START_REF]strain new to humans[END_REF], P [START_REF] Okada | Another novel strain, 261 116E-like G9P[END_REF], and P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] VP4 genes, as well as rare genetic 74 variants of common antigen types, such as G3 VP7 genes of porcine, feline, or lapine origin 75 or genetically diverse porcine-like P[6] or feline-like P[9] VP4 genes [START_REF] De Grazia | Unusual assortment of segments in 2 rare human rotavirus genomes[END_REF]76 Bányai et al., 200376 Bányai et al., , 2009a;;2009b, 2009c;[START_REF] Matthijnssens | Full genomic analysis of human rotavirus strain 406 B4106 and lapine rotavirus strain 30/96 provides evidence for interspecies transmission[END_REF]Matthijnssens et al., , 2009;;[START_REF] Esona | Novel human rotavirus genotype G5P[7] from child with diarrhea, 351 Cameroon[END_REF]77 Martella et al., 200677 Martella et al., , 2008;;[START_REF] Khamrin | Molecular 374 characterization of a rare G3P[3] human rotavirus reassortant strain reveals evidence for 375 multiple human-animal interspecies transmissions[END_REF][START_REF] Chitambar | Molecular characterization of a rare G1P[19] 323 rotavirus strain from India: evidence of reassortment between human and porcine 324 rotavirus strains[END_REF][START_REF] Steyer | Human, porcine and bovine 441 rotaviruses in Slovenia: evidence of interspecies transmission and genome reassortment[END_REF]. 78

Based on sequence similarities, recently a new classification system has been 79

proposed, which designates independent genotypes for each genome segment [Matthijnssens 80 et al., 2008a[Matthijnssens 80 et al., , 2008b]]. This system is being appreciably adopted by the scientific community 81 and several research groups initiated partial or whole genome based strain characterization 82 studies to extend information on features of circulating rotavirus strains. For example, among 83 the 9 non-serotype genes, the VP6 and NSP4 genes have been studied extensively due to their 84 recognized relative genetic linkage and their potential use in determining the original host 85 species for unusual strains with suspected heterologous origin [START_REF] De Grazia | Canine-origin G3P[3] rotavirus strain in child with acute gastroenteritis[END_REF], 2008, 86 2009;[START_REF] Chitambar | Molecular characterization of a rare G1P[19] 323 rotavirus strain from India: evidence of reassortment between human and porcine 324 rotavirus strains[END_REF]Bányai et al., 2009;[START_REF] Khamrin | Molecular characterization of VP4, VP6, VP7, 381 NSP4, and NSP5/6 genes identifies an unusual G3P[10] human rotavirus strain[END_REF][START_REF] Martella | Detection of a porcine-like rotavirus in a child with 403 enteritis in Italy[END_REF]87 Li et al., 2008;Araujo et al., 2007;[START_REF] Khamrin | Molecular 374 characterization of a rare G3P[3] human rotavirus reassortant strain reveals evidence for 375 multiple human-animal interspecies transmissions[END_REF][START_REF] Khamrin | 377 Molecular characterization of rare G3P[9] rotavirus strains isolated from children 378 hospitalized with acute gastroenteritis[END_REF][START_REF] Ghosh | Evidence 368 for independent segregation of the VP6-and NSP4-encoding genes in porcine group A 369 rotavirus G6P[13] strains[END_REF]Iturriza-88 In Taiwan, rotavirus strain surveillance started in 2001 [START_REF] Chen | Sentinel hospital surveillance for rotavirus diarrhea in 321 Taiwan, 2001-2003[END_REF]. Studies 90 have identified the globally common strains, although the predominant antigen types seem to 91 change over time. We have recently reported strain prevalence data over a 3-year period from 92 3 sentinel hospitals located in northern, central and southern Taiwan [START_REF] Wu | Hospital-based surveillance and molecular epidemiology of rotavirus 454 infection in Taiwan, 2005-2007[END_REF]. That 93 study utilized a nucleotide sequence-based genotyping assay, in which type common PCR 94 primers were employed to amplify full-length or partial outer capsid genes. In total 829 95 strains were genotyped. Among these strains we identified G1P [START_REF]strains detected exclusively on the eastern coast of the USA represents 253 the first documented evidence for human infection with G9 strains and could have caused a 254 minor local outbreak[END_REF] strains to be predominant, 96 followed by G3P [START_REF]strains detected exclusively on the eastern coast of the USA represents 253 the first documented evidence for human infection with G9 strains and could have caused a 254 minor local outbreak[END_REF], G9P [START_REF]strains detected exclusively on the eastern coast of the USA represents 253 the first documented evidence for human infection with G9 strains and could have caused a 254 minor local outbreak[END_REF] and G2P[4] strains as additional medically important strains, 97 while 11 further antigen combinations represented minority strains. Unexpectedly, among 98 these minority combinations we detected 2 P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains associated with a G3 and a G9 VP7 99 gene. In subsequent extended surveillance, we detected 3 additional P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains in 2008 and 100 2009 seasons; 2 associated with G3 and 1 with G5 VP7 gene. In an attempt to better 101 understand the origin and relationship of these strains to one another and the few hitherto 102 characterized human and animal P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] instructions. The extracted RNAs were used as the template for RT-PCR with random primers 124 [START_REF] Wu | Hospital-based surveillance and molecular epidemiology of rotavirus 454 infection in Taiwan, 2005-2007[END_REF]. Viral VP4, VP6, VP7 and NSP4 genes were amplified with primers sets 125 Con3/Con2, JRG7/JRG8 or GEN-VP6F/GEN-VP6R, Beg9-End9, and JRG30/JRG31 as 126 described [START_REF] Gentsch | 359 Identification of group A rotavirus gene 4 types by polymerase chain reaction[END_REF][START_REF] Luan Le | Development and characterization of candidate rotavirus 392 vaccine strains derived from children with diarrhoea in Vietnam[END_REF][START_REF] Matthijnssens | Full genomic analysis of human rotavirus strain 406 B4106 and lapine rotavirus strain 30/96 provides evidence for interspecies transmission[END_REF]Gouvea et al, 1990;127 Esona et al;[START_REF] Esona | However, G5 strains had not previously been reported in 243 Taiwan[END_REF]. Viral RNA samples were also analyzed by a 10% polyacrylamide gel and 128 silver staining according to the manufacturer's protocol (Bio-Rad, Hercules, CA, USA). 129 130Sequencing and phylogenetic analysis 131 PCR amplicons were directly sequenced with primers sets Con3/Con2, JRG7/JRG8 or GEN-132 VP6F/GEN-VP6R, Beg9-End9, and JRG30/JRG31 on a ABI 3130 sequencer (Applied 133 Biosystems, CA, US). Rotavirus genotypes were determined using the RotaC software [Maes 134 et al., 2009]. Multiple nucleotide sequences were aligned and the phylogenetic analysis was 135 performed using MEGA 4.0 software [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics 444 Analysis (MEGA) software version 4.0[END_REF]. 136 (750 bp and 250 aa), these strains shared 91.5%-96.2% (nt) and 93.7-97.6% (aa) identities 141 among each other and a lower similarity to 16 other P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains detected in mainland Asia 142 (range, nt, 83.5-86.6%; aa, 89.4-94.1%). In our phylogenetic analysis, both nt (Fig. 1A) and 143 aa (not shown) trees demonstrated that Taiwanese P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains fell into a discrete genetic 144 lineage. An aa alignment of the VP8* fragment revealed 5 aa substitutions in the VP8* 145 fragment of the protein, which were unique for the Taiwanese P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains (Fig. 1B). 146 147 Genetic analysis of the VP7 genes. As determined by nucleotide sequence based genotyping, 148 the 5 Taiwanese P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains exhibited 3 different VP7 specificities: G3 (03-98s185, 07-149 94s126, 07-97s684), G5 (04-97s51), and G9 (07-96s1118) (Fig. 2). 150

The full or nearly full length coding region was determined for the G3 strains. These 3 151 strains shared 92.8% -93.4% nt (and 96.3% -97.5% aa) similarities along a 972 bp long 152 fragment to each other, and a lesser similarity to 183 reference G3 strains (nt, < 90.1%; aa, < 153 95.6%). They are most closely related to porcine isolates (e.g., CP-1). Along the antigenic 154 region these 3 Taiwanese strains shared high similarities, although 1 to 4 aa substitutions were 155 seen (Fig. 3). Phylogenetic analysis found that these 3 strains clustered on a separate branch, 156 distinct from other strains suggesting they represent a new lineage of the G3 VP7 gene (Fig. 157 2). 158

Identification of a G5P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strain in our sample collection was unexpected, as this 159 antigen combination was not identified previously in humans. Sequence analysis of a 828 bp 160 stretch of the VP7 gene of strain 04-97s51 revealed moderate sequence identity to 98 161 reference G5 strains of both animal and human origin (range: nt, 82.2-87.3%; aa, 87.0-162 93.1%). Similar divergence was seen in the neutralization epitopes between 04-97s51 and 163 selected G5 strains. Overall, these similarity ranges together with results of phylogenetic 164 (Fig. 2). 166 A fifth Taiwanese P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strain contained a G9 VP7 gene. Because this specificity was 167 seen in both human and porcine strains in South-East Asia, it was interesting to see whether 168 the Taiwanese isolate clustered with these human or animal rotavirus strains. Of note, the 07-169 96s1118 strain displayed limited sequence similarity with other G9 strains detected in Taiwan 170 or other parts of the world. Overall, this Taiwanese G9 strain shared ≤92.0% nt (and ≤95.7% 171 aa) identity to any known G9 VP7 gene along the full coding region, including both human 172 and porcine strains, and shared particularly lower similarity to the globally emerging variant 173 of G9 VP7 gene (nt, <90%). The closest relatives were among the unique US OM46-like G9 174 strains that were detected during 1997-1998 in Omaha (nt, 92%; aa, 95.1%). The 175 neutralization epitopes of this novel Taiwanese G9 strain also suggested extensive divergence 176 from other G9 strains. All these data demonstrated that 07-96s1118 is not only highly 177 divergent from the globally emerging variant of G9 VP7 gene, but also divergent from all 178 other G9 VP7 genes identified in surveillance studies over the last 30 years (Fig. 2,3). 179 180 Genetic analysis of the VP6 and NSP4 genes. The full or partial coding regions of the VP6 181 and NSP4 genes were determined to further characterize the 5 identified Taiwanese P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] 182 strains.For the VP6 gene, the RotaC [START_REF] Maes | RotaC: a web-based tool for the 395 complete genome classification of group A rotaviruses[END_REF] online genotyping software that was 183 developed to predict rotavirus A genotype specificities identified 2 VP6 genotypes among 184

Taiwanese P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains (Fig. 4). Four strains were classified as genotype I1, a common 185 human VP6 specificity, while 07-96s1118, the unique Taiwanese G9P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strain shared only 186 moderate sequence identity to other genotypes. The automated genotyping platform 187 recommended validating the VP6 gene specificity of this strain through the RCWG, whose 188 opinion was that this gene falls within the newly described I12 VP6 genotype. This genotype 189 KTM368, and the Taiwanese G9P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strain, 07-96s1118. Phylogenetic analysis suggested 191 this genotype is intermediate between genotype I1 (≤85%) and I5 VP6 (≤86%) genotypes and 192 even its representative strains share only marginal nt identities to one another (87.6% nt). The 193 other 4 P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains shared ~90-98% nt identities among each other, and were most closely 194 related to 2 porcine strains (i.e., 04-97s51-like P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains vs. CN86, 97.8-97.9% nt 195 identities and 03-98s185 vs. JL94, 91.1% nt identity). 196 RotaC clustered all five Taiwanese P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains into a single E1 NSP4 genotype with 197 ≤93.4% identity to reference strains (Fig. 4). The P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains shared 93.0%-96.0% nt 198 similarities among one another. Among the reference strains, the rare human-porcine 199 reassortant strains (e.g., LL4260) were most closely related to these Taiwanese P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains 200 (range: nt, 91.7%-93.4%). 201 Three of the 5 Taiwanese P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] rotavirus strains with adequate amount of RNA were 202 further examined for their RNA profiles by polyacrylamide gel electrophoresis (Fig. 5). The 2 203 strains with G5P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] and G9P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] genotypes had long RNA electropherotypes similar to that 204 of the reference Wa strain, whereas one G3P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strain, 03-98s185, had a super-short 205 electropherotype as indicated by its rearranged gene segment 11. 206 207 Discussion 208

This study reports new findings of unusual P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] rotavirus strains from our sentinel hospital 209 based-surveillance during 2004 to 2009. Among 1183 strains G and P typed, there were 5 210 isolates (0.4% of total) belonging to genotype P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF]. These isolates were identified from 211 specimens collected from different years, one in 2005 (0.8%), one in 2007 (0.3%), two in 212 2008 (1%) and one in 2009 (0.6%). Thus, these rare strains were circulating among humans or 213 alternatively in an animal host in Taiwan during most of the surveillance period. 214 There have been a few reports on rotavirus P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strain detection among animals and detection of the virus in humans from Thailand [START_REF] Urasawa | Antigenic and genetic analyses of 447 human rotaviruses in Chiang Mai, Thailand: evidence for a close relationship between 448 human and animal rotaviruses[END_REF], India [Varghese et al., 218 2004] and Vietnam [START_REF] Nguyen | 430 Use of sequence analysis of the VP4 gene to classify recent Vietnamese rotavirus isolates[END_REF]. In the present study, phylogenetic analysis revealed 219 that the Taiwanese strains were genetically closely related to each other but were distinct from 220 human or animal strains from other countries. For example, in the VP8* fragment of VP4 221 there were 5 conserved aa substitutions found in Taiwanese P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains compared to human 222 or animal P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains from other countries. In addition, 4 other aa sites, positions 15, 44, 73 223 and 254, are more closely related to the prototype 4F strain than human or animal P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] 224 strains isolated from other Asian countries. 225

The most prevalent VP7 genotypes in Taiwan were G1 (48.3%), G3 (24.5%), G9 226 (14.1%), G2 (8.0%) and some rare genotypes like G4, G5 and G8 [START_REF] Wu | Hospital-based surveillance and molecular epidemiology of rotavirus 454 infection in Taiwan, 2005-2007[END_REF]. In most 227 studies globally, G1 represented appoximately 50-65% of the strains, followed by G3, G4, 228 and G2 [Gentsch et al., 2005;Santos and Hoshino, 2005]. In addition, an increasing number 229 of rare genotypes have been described recently, many of which have strong genetic 230 relationships with animal rotaviruses [Matthijnssens et al., 2009]. 231 Among the G3 strains, we found 3 with P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] specificity, one each from 2005, 2008 232 and 2009. The nucleotide sequences of these 3 strains showed only moderate similarity 233 between them (only 92.8% -93.4%), and less similarity to 183 reference G3 strains. The most 234 closely related strain was a porcine isolate, CP-1 which clustered on a distinct branch by 235 phylogenetic analysis. Demonstration that there were several amino-acid substitutions in the 236 antigenic sites of these Taiwan G3 strains supports the proposal that they represent a new G3 237 Mc345-like strains. This variant of G9 strains that subsequently spread worldwide to become 265 the 5 th most common strain during a 10-year period are additional examples of introductions 266 of G9 VP7 into human populations [Gentsch et al., 2005;Matthijnssens et al., 2009]. 267

Additional emergences seen in central region of the USA during mid-1990s by a divergent 268 G9P [START_REF]strains detected exclusively on the eastern coast of the USA represents 253 the first documented evidence for human infection with G9 strains and could have caused a 254 minor local outbreak[END_REF] strain (OM46-like) [START_REF] Laird | Characterization of 384 serotype G9 rotavirus strains isolated in the United States and India from 1993 to 2001[END_REF], and other G9 strains (97'SZ37 and T203) with 269 divergent VP7 genes were reported in far-east Asia [Xu et al., GenBank record;Dong et al., 270 2008]. While to date only the lineage III G9 strains have been proven to possess significant 271 epidemiologic potential, the data on a new G9 VP7 gene variant from Taiwan clearly 272 demonstrates that interspecies transmission events are more frequent and the G9 VP7 gene is 273 genetically more divergent than previously thought based on a limited number of gene 274 sequences. 275 Overall, our findings demonstrate that the P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains circulating in Taiwan are 276 different from those in other Asian countries. The distinct evolutionary patterns of these 277 strains may be explained by Taiwan's geographic separation from the rest of Asia by the 278 Pacific Ocean and consequently minimal genetic contact with rotavirus strains of suspect 279 animal reservoirs from other countries. Since P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains may have originated in porcine 280 species, it will be important to conduct surveillance and determine whether similar strains are 281 also present among pigs in Taiwan. 282 indicate the Taiwanese G3P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF], G5P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF], and G9P [START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF]

  -75 nm in diameter and contain 11 genomic segments of double-stranded 65

137 Results 138

 138 Genetic analysis of the VP4 genes. A 831 bp fragment of the VP4 gene (corresponding to nt 139 34-864, strain Mc345) of 5 Taiwanese P[START_REF] Strain | Molecular features of the G5 VP7 gene of our Taiwanese strain belonged to a 245 phylogenetic lineage distinct from those represented by other strains. 246 Unlike the G5P[END_REF] strains was sequenced. Along a shorter stretch 140

  this Taiwanese G5 strain fell into a new lineage of the G5 VP7 gene 165

  by 2 strains, the reference human Bangladeshi G11P[25] strain, 190

  first detected in South Asia and genetically it was most closely related to 264

  Figure legends 456

Fig. 4 .

 4 Fig. 2. Nucleotide sequence based phylogenetic trees of the G3, G5 and G9 VP7 genes. The 465

Fig

  Fig. 2.

Fig

  Fig. 4.

106 Materials and Methods 107

	115	Laboratory testing
	116	Stool specimens were collected and screened for rotavirus VP6 antigen using an EIA kit
	117	(RIDASCREEN® Rotavirus, R-Biopharm AG, Germany) at each hospital. Rotavirus EIA-
	118	positive specimens were shipped to Rotavirus Reference Laboratory at Taiwan CDC for G-
	119	and P-genotyping.
	120
	121	RT-PCR and Polyacrylamide Gel Electrophoresis
	122 123	Viral RNA was extracted from 10% (wt/v) fecal supernatants by MagNA Pure LC DNA F r o isolation kit (Roche Diagnostics GmbH, Germany) according to the manufacturer's
		P
		e
		e r
	103 104	strains, we determined and analyzed the full length or nearly full length VP7 genes, the variable VP8* fragment of the VP4 gene, and the genotype R
	105	diversity and potential genetic linkage among the NSP4 and the VP6 genes. e v i e
	108	Case definition and data collection	w
	109	From January 2005 to December 2009, a total of 6,346 stool specimens were collected from
	110	children less than 5 years old who were hospitalized for treatment of acute gastroenteritis
	111	(AGE) in 3 sentinel hospitals located in northern, central and southern Taiwan. AGE was
	112	defined as three or more episodes of watery diarrhoea or looser-than-normal stool in the 24 h
	113	before presentation.
	114
			6

humans since 1990, beginning with the first report of the isolation and characterization of

P[19] prototype strain 4F in 1994[19] prototype strain 4F in [Burke et al., 1994]]. Several other studies document

regionally common strain in Brazil during 2004[START_REF] Alfieri | 297 Characterization of human rotavirus genotype P[8]G5 from Brazil by probe-hybridization 298 and sequence[END_REF]. Unusual human G5P[6]

rotaviruses were identified from AGE children under 5 in China and Vietnam[Ahmed et al.,

differing in growth in vitro and in pathogenicity: distinct VP4 sequences and conservation

of NS53, VP6 and VP7 genes. J Gen Virol 75:2205-2212.

De Grazia S, Giammanco GM, Martella V, Ramirez S, Colomba C, Cascio A, Arista S. 2008.

children with diarrhea in Italy. J Clin Microbiol 46:357-360.

Gentsch JR, Laird AR, Bielfelt B, Griffin DD, Banyai K, Ramachandran M, Jain V, Cunliffe

Matthijnssens J, Ciarlet M, Heiman E, Arijs I, Delbeke T, McDonald SM, Palombo EA,

Parashar UD, Gibson CJ, Breese JS, Glass RI. 2006. Rotavirus and severe childhood diarrhea.

Emerg Infect Dis 12:304-306.

John Wiley & SonsJournal of Medical Virology

Acknowledgements

This study was financially supported in part by research grant of DOH97-DC-1102 and 285 "National Research Program for Genome Medicine" (supported grant: 94-0324-19-F-01-00-286 from Centers for Disease Control, 287 Department of Health, Execute Yuan, Taiwan. K. B. was supported by the Hungarian 288


Martin G Kees
email: martin.kees@charite.de

Justus W Hilpert

Carsten Gnewuch

Frieder Kees

Stephan Voegeler

Clearance of vancomycin during continuous infusion in Intensive Care Unit patients: correlation with measured and estimated creatinine clearance and serum cystatin C

Keywords:

195 mL/min) and its correlation with measured creatinine clearance (CL Crea ), estimated creatinine clearance using the Cockcroft-Gault formula (CL CG ) and estimated glomerular filtration rate according to Hoek's formula based on serum cystatin C (GFR Hoek ) was investigated. The correlation between CL Van and CL Crea was very good (r 2 = 0.88), but it was rather poor with CL CG (r 2 = 0.37) and was acceptable with GFR Hoek (r 2 = 0.70). For VAN dose adjustments in ICU patients, determination of cystatin C may be an interesting and practical alternative to measured CL Crea , whereas the Cockcroft-Gault formula should be used with caution.

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

Introduction

Vancomycin (VAN) is still the standard treatment for infections caused by meticillinresistant Staphylococcus aureus (MRSA), despite the introduction of some alternative agents [START_REF] Thoracic | Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia[END_REF][START_REF] Tunkel | Practice guidelines for the management of bacterial meningitis[END_REF]. However, the narrow therapeutic range and a trend towards higher minimum inhibitory concentrations (MICs) make precise dosing of VAN preferable for successful treatment and, for this purpose, sound population-specific pharmacokinetic data are required [START_REF] Rybak | Therapeutic monitoring of vancomycin in adult patients: a consensus review of the American Society of Health-System Pharmacists, the Infectious Diseases Society of America, and the Society of Infectious Diseases Pharmacists[END_REF].

VAN is predominantly eliminated via the kidney and the maintenance dose is mainly determined by the glomerular filtration rate (GFR) [START_REF] Moellering | Pharmacokinetics of vancomycin[END_REF]. The GFR can be determined by various methods, the validity of which are typically proportional to effort. Whereas clearance of exogenous substances such as inulin is the suggested gold standard, measured creatinine clearance can be considered as the clinical reference standard.

Assuming constant and predictable production of creatinine, its clearance can be estimated from serum creatinine alone, e.g. using the popular Cockcroft-Gault formula [START_REF] Cockcroft | Prediction of creatinine clearance from serum creatinine[END_REF]. By analogy, serum cystatin C has been established as an indicator of GFR [START_REF] Dharnidharka | Serum cystatin C is superior to serum creatinine as a marker of kidney function: a meta-analysis[END_REF].

Surprisingly, there are only few pharmacokinetic studies on VAN in Intensive Care Unit (ICU) patients without renal replacement therapy. Only one study used a classic prospective pharmacokinetic design [START_REF] Polard | Non steady state and steady state PKS Bayesian forecasting and vancomycin pharmacokinetics in ICU adult patients[END_REF], whereas three other studies retrospectively analysed data from routine therapeutic drug monitoring (TDM) [START_REF] Pea | Prospectively validated dosing nomograms for maximizing the pharmacodynamics of[END_REF][START_REF] Mar | Pharmacokinetic/pharmacodynamic analysis of vancomycin in ICU patients[END_REF][START_REF] Llopis-Salvia | Population pharmacokinetic parameters of vancomycin in critically ill patients[END_REF]. These studies exclusively used estimations based on serum creatinine as a measure of renal function, although the underlying assumptions are particularly questionable in critically ill patients [START_REF] Hoste | Assessment of renal function in recently admitted critically ill patients with normal serum creatinine[END_REF][START_REF] Herrera-Gutiérrez | Replacement of 24-h creatinine clearance by 2-h creatinine clearance in intensive care unit patients: a singlecenter study[END_REF][START_REF] Conil | Assessment of renal function in clinical practice at the bedside of burn patients[END_REF][START_REF] Cherry | Accuracy of short-duration creatinine clearance determinations in predicting 24-hour creatinine clearance in critically ill and injured patients[END_REF][START_REF] Martin | Pitfalls of using estimations of glomerular filtration rate in an intensive care population[END_REF][START_REF] Poggio | Performance of the Cockcroft-Gault and Modification of Diet in Renal Disease equations in estimating GFR in ill hospitalized patients[END_REF]. Consequently, in these studies a large part of the Although clinical superiority in terms of outcome has not been demonstrated, there is substantial theoretical support for continuous infusion of VAN [17]. In the surgical ICU at Charité University Hospital, Campus Benjamin Franklin (Berlin, Germany), continuous infusion is applied as the standard administration form because it simplifies interpretation of TDM and dose adjustments. As an ancillary effect, continuous infusion permits easy determination of drug clearance. At steady state, determination of a single plasma concentration would suffice for calculation of clearance.

The aim of this study was to determine VAN clearance (CL Van ) in ICU patients during continuous infusion and to investigate its relationships with measured creatinine clearance (CL Crea ), estimated creatinine clearance using the Cockcroft-Gault formula (CL CG ) and estimated GFR according to Hoek's formula based on serum cystatin C (GFR Hoek ), as a basis for dosing recommendations.

Patients and methods

Study design and patients

This prospective, single-centre study was approved by the Institutional Ethics Committee of Charité University Hospital. Adult patients in the 22-bed surgical ICU who received treatment with VAN continuous infusion were enrolled. Patients A c c e p t e d M a n u s c r i p t undergoing renal replacement therapy were excluded. Written informed consent was obtained from either the patient or their legal representative.

Vancomycin dosage and therapeutic drug monitoring

The attending physician started VAN treatment when infection by a susceptible pathogen was proven or suspected. According to the internal protocol, a loading dose was administered as a 1-h infusion, followed by continuous infusion of VAN. The initial infusion rate was chosen according to the estimated creatinine clearance.

Daily at 05:00h, TDM was performed and the infusion rate was adjusted to a target concentration between 20 mg/L and 25 mg/L.

Pharmacokinetic calculations

As soon as routine TDM showed the VAN serum concentration to be within the desired range and the infusion rate had not been changed for more than 24 h, blood was sampled from arterial catheters at 11:00h, 17:00h, 23:00h and 05:00h. Urine was collected quantitatively in three 6-h fractions from 11:00h to 05:00h. VAN [START_REF] Beckmann | Tissue concentrations of vancomycin and moxifloxacin in periprosthetic infection in rats[END_REF]. A reversed-phase column (Gemini-NX C18, 3 m, internal diameter 15  0.46 cm; Phenomenex, Aschaffenburg, Germany) was used for separation with an eluent composed of 1000 mL of 50 mM sodium dihydrogen phosphate and 100 mL of acetonitrile. The pH was adjusted to 3.9 with o-phosphoric acid. Interference by concomitant medication in a few patients could be avoided by adjusting the pH between 3.9 and 3.1, as the retention time of VAN is sensitive to pH. Based on spiked quality control samples, intra-and inter-assay imprecision as well as bias were <5%.

Concentrations of creatinine were determined enzymatically using an ADVIA Chemistry System (ECRE 2; Siemens Healthcare Diagnostics, Eschborn, Germany).

Concentrations of cystatin C were determined using a BN™ II Nephelometer (Siemens Healthcare Diagnostics).

Statistical analysis

SPSS 17 for Mac OS X (SPSS Inc., Chicago, IL) was used for statistical analysis.

Parametric linear regression was used to evaluate correlations between parameters. Two-sided  errors <0.05 were considered statistically significant and were reported as P-values. Data are presented as median (range) or mean (95% confidence interval), as appropriate.

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

Results

A total of 25 patients (14 male, 11 female) were enrolled in the study (Table 1). Most commonly, sampling was performed around Day 5 (range, Day 2-15) of treatment with VAN. In one patient, only one 6-h interval (two serum samples and one urine fraction) was available because VAN treatment was terminated. The concentrations were sufficiently constant to assume steady-state conditions in 24 patients, whereas in 1 patient a continuous decline of 32% over 18 h was observed, probably due to acute arterial hypertension with enhanced glomerular filtration. In this patient, only the last concentration, which is closest to steady-state, was used to calculate CL Van .

The study population exhibited a wide range of renal function, with a median CL Crea of 106 mL/min (range 25-195 mL/min), providing an excellent basis to assess the relationship between CL Van and renal function. Fig. 1 depicts the relationships between CL Van with CL Crea , CL CG and GFR Hoek . The respective regression equations and coefficients of determination are shown in Table 2.

Discussion

The present study compares measured creatinine clearance (CL Crea ), creatinine clearance estimated by the Cockcroft-Gault formula from serum creatinine (CL CG ) and GFR estimated by the Hoek formula from serum cystatin C (GFR Hoek ) as determinants of CL Van in ICU patients.

As could be expected, CL Crea showed the closest correlation with CL Van with a residual error (1-r 2 ) of only ca. 12%, which includes measurement errors and further Gault formula estimates creatinine production from age, weight and sex, whereas the Hoek formula assumes cystatin C production to be proportional to BSA. Catabolism, immobilisation and other disturbances of homeostasis negatively affect the precision of these formulas in ICU patients, as has been documented by a number of previous studies [START_REF] Hoste | Assessment of renal function in recently admitted critically ill patients with normal serum creatinine[END_REF][START_REF] Herrera-Gutiérrez | Replacement of 24-h creatinine clearance by 2-h creatinine clearance in intensive care unit patients: a singlecenter study[END_REF][START_REF] Conil | Assessment of renal function in clinical practice at the bedside of burn patients[END_REF][START_REF] Cherry | Accuracy of short-duration creatinine clearance determinations in predicting 24-hour creatinine clearance in critically ill and injured patients[END_REF][START_REF] Martin | Pitfalls of using estimations of glomerular filtration rate in an intensive care population[END_REF][START_REF] Poggio | Performance of the Cockcroft-Gault and Modification of Diet in Renal Disease equations in estimating GFR in ill hospitalized patients[END_REF].

In a recent study in Japanese non-ICU patients, GFR Hoek correlated very well with CL Van (r 2 = 0.84) [START_REF] Tanaka | Population pharmacokinetic analysis of vancomycin using serum cystatin C as a marker of renal function[END_REF]. As outlined above, it is not surprising that the formula performs worse in the ICU patients in the current study, but the correlation (r 2 = 0.70) seems acceptable. We would like to emphasise that the Hoek formula estimates GFR, which A c c e p t e d M a n u s c r i p t is systematically lower than creatinine clearance owing to some tubular secretion of creatinine.

Regarding CL CG , although the correlation is statistically highly significant, its value in predicting CL Van in ICU patients seems doubtful. Other studies in ICU patients reported correlations with r 2 between 0.43 and 0.56 [START_REF] Polard | Non steady state and steady state PKS Bayesian forecasting and vancomycin pharmacokinetics in ICU adult patients[END_REF][START_REF] Pea | Prospectively validated dosing nomograms for maximizing the pharmacodynamics of[END_REF][START_REF] Mar | Pharmacokinetic/pharmacodynamic analysis of vancomycin in ICU patients[END_REF][START_REF] Llopis-Salvia | Population pharmacokinetic parameters of vancomycin in critically ill patients[END_REF], which is somewhat better than in the data here (r 2 = 0.37). However, in one study patients who had been in hospital for more than 14 days were excluded [START_REF] Pea | Prospectively validated dosing nomograms for maximizing the pharmacodynamics of[END_REF], and in another study the Cockcroft-Gault formula was modified by truncating values above 120 mL/min [START_REF] Llopis-Salvia | Population pharmacokinetic parameters of vancomycin in critically ill patients[END_REF]. It should be kept in mind that exact values throughout all the pathophysiological range are required for reliable drug dosing, not just the discrimination of normal from abnormal renal function.

Of course, using the Cockcroft-Gault formula is probably the easiest and least expensive way to obtain an estimate of renal function. However, collecting urine in ICU patients is typically performed anyway for calculating fluid balances and seems a small additional effort, which is rewarded by a substantial gain in precision. Several studies suggest that short collection periods of 1-2 h are sufficient for accurate measurements [START_REF] Hoste | Assessment of renal function in recently admitted critically ill patients with normal serum creatinine[END_REF][START_REF] Herrera-Gutiérrez | Replacement of 24-h creatinine clearance by 2-h creatinine clearance in intensive care unit patients: a singlecenter study[END_REF][START_REF] Sladen | Two-hour versus 22-hour creatinine clearance in critically ill patients[END_REF][START_REF] Wilson | The validity of two-hour creatinine clearance studies in critically ill patients[END_REF]. We therefore recommend the use of measured CL Crea for prediction of CL Van in ICU patients.

Our data can be used as a basis to predict dosing requirements. Current recommendations suggest an area under the concentration-time curve from 0-24 h (AUC 24h )/MIC of 400 h as the relevant pharmacokinetic/pharmacodynamic target [START_REF] Rybak | Therapeutic monitoring of vancomycin in adult patients: a consensus review of the American Society of Health-System Pharmacists, the Infectious Diseases Society of America, and the Society of Infectious Diseases Pharmacists[END_REF], which corresponds to a mean serum concentration (C mean ) of 16.7 mg/L for an MIC of A c c e p t e d M a n u s c r i p t 1.0 mg/L. For continuous infusion, target concentrations between 15 mg/L and 25 mg/L (free concentrations of 4-5 MIC) have been suggested [START_REF] Pea | Prospectively validated dosing nomograms for maximizing the pharmacodynamics of[END_REF][START_REF] Vuagnat | High dose vancomycin for osteomyelitis: continuous vs. intermittent infusion[END_REF][START_REF] Wysocki | Continuous versus intermittent infusion of vancomycin in severe staphylococcal infections: prospective multicenter randomized study[END_REF]. The daily maintenance dose (dMD) can be calculated as dMD = 1440 min  C mean  (0.63  CL Crea + 2.8 mL/min), or dMD = AUC 24h  (0.63  CL Crea + 2.8 mL/min). Owing to the linear pharmacokinetics of VAN [26], AUC 24h is independent of the dosing regimen (i.e. it is the same whether the daily dose is given as one, two, four or an infinite number of single doses). Therefore, according to classic pharmacokinetic theory the formula should also be applicable to intermittent infusion. Using the Hoek formula, the expression (0.63  CL Crea + 2.8 mL/min) needs to be replaced by (0.67  GFR Hoek + 3.2 mL/min). We advise against the use of the Cockcroft-Gault formula for calculating dosing requirements.

Finally, some limitations of this study must be mentioned. This study was performed on a surgical ICU in a university hospital. The majority of patients underwent neurosurgery or general surgery. The results should be extrapolated with caution to other populations.

Calculation of CL Van in this study requires the presence of steady state. Long halflives make it difficult to attain the real equilibrium, and even impossible if clinical conditions change as is likely in ICU patients. As the individual concentration-time courses were fairly constant and the number of patients with rising or falling concentrations was well balanced, we consider this error small and not systematic.

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  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 ABSTRACT Vancomycin (VAN) dosing requires adjustment to renal function, which is often estimated using the Cockcroft-Gault formula; however, its precision is poor in Intensive Care Unit (ICU) patients. VAN clearance (CL Van ) during continuous infusion was prospectively determined in 25 ICU patients [14 male, 11 female; age range 31-82 years; body mass index (BMI) 16.5-41.5 kg/m 2 ; Acute Physiology and Chronic Health Evaluation (APACHE) II score at admission 8-36; creatinine clearance 25-

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  c c e p t e d M a n u s c r i p t variance in VAN clearance remains unaccounted for. It is unclear whether this reflects unknown physiological changes during critical illness or the shortcomings of the applied methods.

  and creatinine concentrations were determined in all serum specimens and, owing to budgetary restrictions, cystatin C was determined only in the first and last specimens. Urine specimens were analysed for creatinine. Clearance of VAN (CL Van ) and creatinine clearance (CL Crea ) were calculated as follows: CL Van = infusion rate of VAN/serum concentration of VAN A c c e p t e d M a n u s c r i p t CL Crea = (urine volume  urine concentration of creatinine)/(collection time  serum concentration of creatinine) Creatinine clearance based on serum creatinine (CL CG ) was estimated using the Cockcroft-Gault formula [5]: CL CG (mL/min) = [140age (years)]  weight (kg)/[72  serum creatinine (mg/dL)]  0.85 for women GFR based on serum cystatin C was estimated by the Hoek formula (GFR Hoek ) [18] and the Du Bois formula for body surface area (BSA) [19]: GFR Hoek = [80.35/serum cystatin C (mg/L) -4.32] mL/min/1.73 m 2  BSA BSA (m 2 ) = 0.007184  height (cm) 0.725  weight (kg) 0.4252.4. Analysis of vancomycin, creatinine and cystatin CBlood samples were allowed to clot for 30 min, centrifuged and stored at -20 C. Urine samples were frozen at -20 C without prior treatment. Routine TDM was performed in the hospital's central laboratory (Patients 1 to 13, VANC2, Roche Diagnostics, Mannheim, Germany; and Patients 14 to 25, QMS ® Vancomycin, Thermo Fisher Scientific, Dreieich, Germany). Serum concentrations of VAN for pharmacokinetic calculations were assayed by high-performance liquidA c c e p t e d M a n u s c r i p t chromatography (HPLC) with photometric detection at 240 nm as previously described

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  c c e p t e d M a n u s c r i p t undetected covariates. Apart from analytical errors, pharmacokinetic calculations assume constant physiological conditions throughout the study period, which is probably never true in ICU patients. Thus, it seems unlikely that further research would identify additional relevant covariates of CL Van in comparable populations. In principle, estimation of GFR based on serum concentrations of endogenous markers alone works like the calculation of drug clearance during continuous infusion, as applied in this study for VAN: Clearance = infusion or production rate/serum concentration For ideal markers, which undergo exclusive elimination by free glomerular filtration, clearance would be equal to the GFR, whilst restricted filtration, secretion, reabsorption or non-renal clearance may require corrective factors. The Cockcroft-

Fig. 1 .

 1 Fig. 1. Relationship between clearance of vancomycin (CL Van ) and (A) measured

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 Table 2

 2 Regression equations of the dependence of vancomycin clearance (CL Van ) on measured creatinine clearance (CL Crea ), creatinine clearance estimated using the Cockcroft-Gault formula (CL CG ) and glomerular filtration rate estimated using the Hoek formula (GFR Hoek ). Listed are point estimates (95% confidence interval) of (a) slope and (b)

Table 1

 1 Characteristics of the 25 patients (14 male, 11 female) in the study

	Median Range

Acknowledgments

The authors thank the medical and nursing staff on the surgical ICU at Charité University Hospital, Campus Benjamin Franklin (Berlin, Germany) for their reliable help as well as Dr Karin Hensel-Wiegel, Department of Clinical Chemistry, for her cooperation.

Funding

This work was supported by institutional sources only.

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

The dosing recommendations are derived from descriptive data only. Their predictive performance should be validated in prospective studies with heterogeneous populations.

To summarise, we demonstrated in ICU patients that CL Van is closely correlated with measured CL Crea , which is therefore a good basis for estimating dosing requirements.

Serum cystatin C may be a useful and practical alternative. Use of the Cockcroft-Gault formula bears large uncertainties in this context.

Competing interests

None declared.

Ethical approval

Ethical approval was received from the Institutional Ethics Committee of the Charité University Hospital, Campus Benjamin Franklin (reference no. EA4/113/07).