A R Tacchella

G Natalizia

R Melioli

P Bandettini

M C Pietro

R Diana

Gasparini

Donatella Panatto
email: panatto@unige.it

D Amicizia

R Giacchino

A R Natalizia

G Melioli

R Bandettini

P Di

M C Diana

R Gasparini

BURDEN OF ROTAVIRUS INFECTIONS IN LIGURIA (NORTHERN ITALY): HOSPITALIZATIONS AND POTENTIAL SAVING BY VACCINATION

Keywords: Rotavirus, hospitalisation, vaccination, gastroenteritis

Burden of rotavirus infections in Liguria, northern Italy:

hospitalisations and potential savings by vaccination

Text Introduction

Rotavirus gastroenteritis (RVGE) is a major cause of infantile gastroenteritis worldwide and is responsible for high rates of mortality and morbidity in developing countries, where hygiene conditions and living standards are low; in industrialized countries, it remains a common cause of medical consultations and hospital admissions [START_REF] Parashar | Rotavirus and severe childhood diarrhea[END_REF] [2], indicating that improved sanitation does not reduce the circulation of the virus.

The clinical picture varies considerably and can be serious, comprising severe diarrhoea, electrolyte disorders and dehydration. The risk of hospitalisation is high in children under 5 years old, particularly in the 6-24-month age-group [START_REF] Dennehy | Transmission of rotavirus and other enteric pathogens in the home[END_REF]. Comparison with other agents of gastroenteritis indicates that rotavirus (RV) is more frequently associated with severe symptoms [START_REF] Bernstein | Rotavirus overview[END_REF] [START_REF] Giaquinto | Prospective study of the burden of acute gastroenteritis and rotavirus gastroenteritis in children less than 5 years of age, in Padova, Italy[END_REF].

The infection is passed from person to person through the oro-faecal route, but transmission probably occurs through droplets, too [START_REF] Fischer | Rotavirus vaccines and the prevention of hospital-acquired diarrhea in children[END_REF]. Moreover, the virus is very difficult to eradicate and remains viable on dry surfaces for 1 -10 days and on human hands for at least 4 hours [START_REF] Ansari | Comparison of cloth, paper, and warm air drying in eliminating viruses and bacteria from washed hands[END_REF] [START_REF] Ansari | Survival and vehicular spread of human rotaviruses: possible relation to seasonality of outbreaks[END_REF]. These characteristics can explain why RV is a cause of nosocomial infections [START_REF] Berner | Occurrence and impact of community-acquired and nosocomial rotavirus infections a hospital-based study over 10 years[END_REF].

It has been recognized that diarrhoea remains a leading cause of morbidity among US children, accounting for approximately 10% of all inpatient admissions of US children younger than 5 years of age [START_REF] Bernstein | Rotavirus overview[END_REF] [START_REF] Charles | Hospitalisations associated with rotavirus gastroenteritis in the United States, 1993-2002[END_REF]. Soriano-Gabarrò estimated that Europe has 3.6 million cases per year among children under 5 years of age, 87,000 admissions and about 700,000 medical consultations; however, mortality caused by rotavirus disease is low: about 241 deaths per year [START_REF] Soriano-Gabarró | Burden of rotavirus disease in European Union countries[END_REF].

Two rotavirus vaccines have recently been licensed in Italy. These could be an efficacious means of reducing the burden of the infection and the costs to the healthcare system and society, rendering RV illness a vaccinepreventable disease [START_REF] Guarino | Italian Pediatrician's consensus statement on anti-Rotavirus vaccines[END_REF].

There is no gastroenteritis surveillance system in Italy. Moreover, data on the burden of infection are needed in order to understand the potential benefits of introducing universal rotavirus vaccination in children under 1 year of age. We therefore conducted a retrospective study to evaluate the rates of gastroenteritis admissions to the Emergency Department (ED) and of rotavirus-related hospitalisation in children under 5 years of age. We calculated the number of RV cases that could potentially be avoided through the universal vaccination of children.

Materials and methods

Approval for this study was granted by the Local Ethics Committee.

Study population

A retrospective epidemiological study of admissions to the ED and hospitalisations of children < 5 years with acute gastroenteritis (AGE) was carried out at Giannina Gaslini Children's Hospital in Genoa (Northern Italy).

Giannina Gaslini Hospital is the only paediatric hospital covering the metropolitan area of Genoa, which has 39,176 children aged less than 5 years [START_REF]ISTAT 14th Italian Population Census[END_REF].

The study analyzed epidemiological data from 2006.

Case definition of subjects admitted to the ED Gastroenteritis was defined as the presence of diarrhoea (> 3 watery stools within a period of 24 h) and/or vomiting, with or without fever [START_REF] Coffin | Impact of acute rotavirus gastroenteritis on pediatric outpatient practices in the United States[END_REF]. All subjects recruited met the clinical case definition.

Nosocomial case definition

When the onset of gastroenteritis (presence of diarrhoea > 3 watery stools within a period of 24 h and/or vomiting, with or without fever [START_REF] Coffin | Impact of acute rotavirus gastroenteritis on pediatric outpatient practices in the United States[END_REF]) occurred after three days of hospitalisation, nosocomial infection was assumed. Nosocomial RV infections were included in the study.

Admissions to ED and Hospitalisations

The annual number of admissions to the ED due to gastroenteritis and the hospitalisation rate for gastrointestinal illness were collected by extracting these data from the Data Elaboration Centre. Giannina Gaslini Hospital has a Data Elaboration Centre which registers the diagnosis on admission, triage code and diagnosis on discharge. The medical records of the RV-positive children were collected and analyzed.

To calculate the hospitalisation rate due to RV, the virus was sought in the faeces of children hospitalised for acute gastroenteritis.

Laboratory assay

The samples of faeces collected from hospitalised children were analyzed by means of rapid immunochromatographic assay (Combi-strip Coris BioConcept, Belgium) in order to detect RV.

Evaluation of the cases potentially avoided by universal vaccination

We calculated the number of admissions to the ED, admissions to Short Observation (SO) and hospitalisations that could be avoided by universal RV vaccination. Subjects with severe AGE who declined hospitalisation were not considered in the calculation of cases potentially avoided by vaccination.

In order to calculate the potentially avoided cases, it is essential to know the severity of the disease, as the efficacy of the RV vaccine varies according to disease severity [START_REF] Vesikari | Efficacy of human rotavirus vaccine against rotavirus gastroenteritis during the first 2 years of life in European infants: randomised, double-blind controlled study[END_REF]. In hospitalised children, disease severity was graded by means of the 20-point Vesikari scale [START_REF] Ruuska | Rotavirus disease in Finnish children: use of numerical scores for clinical severity of diarrhoeal episodes[END_REF]. As it was not possible to score disease severity in subjects in either the ED or the SO, these cases were regarded as moderate, in accordance with the literature [START_REF] Vesikari | Efficacy of human rotavirus vaccine against rotavirus gastroenteritis during the first 2 years of life in European infants: randomised, double-blind controlled study[END_REF].

As stool samples from children examined in the ED or kept in SO are not routinely collected, RV positivity was estimated by using data from the literature, particularly the results obtained by Forster [START_REF] Forster | Hospital-based surveillance to estimate the burden of rotavirus gastroenteritis among European children younger than 5 years of age[END_REF].

The parameters used in the analysis of the potentially avoided cases in the basic case and in the sensitivity analysis range are reported in Table 1. We performed separate sensitivity analyses for the following parameters: variation in RV-positive cases in the ED, in SO and in hospitalisation with community-acquired RV infection, vaccine efficacy and coverage. It was not possible to calculate the rate of nosocomial RV infections, as we do not know the denominator, namely the total number of cases of nosocomial gastroenteritis.

The potentially avoided cases were calculated by means of the following formula:

A = N * E * C
Where A = number avoided cases; N = number of RV-positive cases; E = vaccine efficacy; C = coverage rate.

Cost evaluation

The costs associated to emergency room admission for gastroenteritis, hospitalisation and nosocomial RV infection were estimated by using Gaslini Hospital's tariffs (June 30, 2006).

The cost of emergency room admission was estimated as € 141 ($ 180.30). The cost of SO admission was estimated as € 1000 ($ 1278.77). The DRG tariffs associated to all hospitalisations due to RV disease were analysed and the average cost was fixed at € 1478 ($ 1890.02). The cost of nosocomial RV infection was estimated as € 2000 ($ 2557.54), considering the average length of hospitalisation.

Statistical analysis

Statistical analysis was performed by means of the SPSS 15.0 statistical package for Windows (SPSS, Chicago USA). We estimated the Relative Risk by Odds Ratio calculation by means of Fisher's exact test. The level of significance adopted was p<0.05. Quantitative variables were expressed as means, medians and standard deviations (SD), and qualitative variables as frequency and percentage with 95% confidence intervals.

Excel (Microsoft) software was used for sensitivity analyses.

Results

In 2006, total admissions to the paediatric ED were 20,690; total admissions to the ED due to gastroenteritis numbered 2396 (11.58% of total admissions, 95% CI 10. [START_REF]Word Health Organization (WHO)[END_REF] [START_REF] Ruuska | Rotavirus disease in Finnish children: use of numerical scores for clinical severity of diarrhoeal episodes[END_REF]; on the basis of disease severity, the number of cases avoided through vaccination was computed by taking into account the different efficacy of the vaccine.

The mean Vesikari score in patients with community-acquired RV infections was 12.93 (range 9-20); 185 (85.6%) patients had a score > 11, which is generally regarded as indicating severe disease. In patients with nosocomial infections, the mean Vesikari score was 12.41 (range 6-16) and 18 (82%) had a score > 11.

We also registered the main and secondary discharge diagnoses in children hospitalised with communityacquired RV infection. Briefly, with regard to the main diagnosis on discharge, an ICD00861 code (Rotavirus intestinal infection) was assigned to 50 patients ( The number of RVGE cases was highest towards the end of winter and at the beginning of spring, though cases were diagnosed throughout the year; a clear predominance was seen during February, March and April.

Figure 1 A shows the variation in the number of cases potentially avoided through universal RV vaccination on varying the number of RV-positive cases. Considering the base case, 71 cases examined in the ED, 332 cases in the SO unit and 176 hospitalised cases could be avoided (corresponding to 45 hospitalizations per 10000 children aged less than 5 years in Genoa in 2006). Figure 1 B also shows the variation in the number of cases avoided on the basis of the efficacy of the RV vaccine. Figure 1 C shows the variation in the number of cases avoided on the basis of the coverage rate of RV vaccination. Considering the base case, the cost of RVGE was calculated as € 800,079. The universal vaccination of the children under 1 year of age living in the study area (6,529 subjects) would cost the healthcare system a maximum of € 457,030 (€ 70,00 per vaccination cycle), considering a 100% coverage rate.

Discussion

The availability of RV vaccines has prompted many researchers to evaluate the epidemiological and economic burden of RV disease. In Italy, however, it is difficult to make an accurate assessment of the incidence of RVGE, as no surveillance system is in place.

The results of our study confirm that acute gastroenteritis, particularly RVGE, is a major cause of ED admission and hospitalisation among children below 5 years of age. The disease burden was seen to be heavy and similar to the percentages reported in other Italian studies [17] [18]. Giaquinto et al. [START_REF] Giaquinto | Prospective study of the burden of acute gastroenteritis and rotavirus gastroenteritis in children less than 5 years of age, in Padova, Italy[END_REF] reported a heavier impact; they found that 40% of acute enteritis cases in children under 5 years were caused by RV, the virus being responsible for 68% of hospitalisations, 61% of ED examinations and 33% of cases of primary care in the Padua area (Northern Italy). Direct comparison is difficult, owing to differences in study designs, laboratory tests and study periods. It should be noted that the magnitude of RV epidemics varies from year to year; indeed, in our study period (2006) the circulation of RV was low. Comparison of our findings with those of the latest European studies reveals a different RV burden on ED admissions and hospitalisations. This variability can be attributed to many factors, such as different policies of health-system access and cultural factors that influence the tendency to seek healthcare [START_REF] Forster | Hospital-based surveillance to estimate the burden of rotavirus gastroenteritis among European children younger than 5 years of age[END_REF] [START_REF] López-De-Andrés | Hospitalisations associated with rotavirus gastroenteritis in Spain, 2001-2005[END_REF].

In our study, the mean hospital stay among children with community-acquired RV was 4.18 days; this is similar to the European mean [START_REF] Forster | Hospital-based surveillance to estimate the burden of rotavirus gastroenteritis among European children younger than 5 years of age[END_REF] [20] [START_REF] Wildi-Runge | A 4-year study on clinical characteristics of children hospitalised with rotavirus gastroenteritis[END_REF]. The age-class at greatest risk of hospitalisation for community-acquired RVGE was 0-23 months, while the distribution of nosocomial cases was similar in all age-classes.

Complications were rare and no deaths due to RV disease occurred. However, the Vesikari scores revealed that 85.6% of hospitalised patients with community-acquired infection suffered severe disease and received fluid infusion.

Our data show that RV coded discharges capture only a part of all RV hospitalisations. Other authors [START_REF] Marsella | Epidemiology of rotavirus-associated hospital admissions in the province of Ferrara, Italy[END_REF] [START_REF] Marchetti | Monitoring the rate of hospitalisation before rotavirus immunization in Italy utilizing ICD9-CM regional databases[END_REF] have confirmed this finding. Physicians recorded the ICD code 00861 on the main discharge diagnosis form in only 23.14% of RV-positive cases with community-acquired infection, and on the secondary discharge diagnosis form in 42.12%; the RV code was attributed to only 65.26% of RV-positive cases. Comparison with the data from the discharge diagnosis database alone, which is the most easily accessible official data source on hospitalisation, reveals that RV-associated hospitalisation is underestimated. Laboratory data indicated that the burden on RV hospitalisation was close to reality.

A clear seasonal distribution emerged, with the greatest incidence occurring at the end of winter and at the beginning of spring (between March and April); a lower circulation was registered during the other months of the year. These data are similar to those of other studies [START_REF] López-De-Andrés | Hospitalisations associated with rotavirus gastroenteritis in Spain, 2001-2005[END_REF] [21] [START_REF] Rendi-Wagner | Active hospital-based surveillance of rotavirus diarrhea in Austrian children, period 1997 to 2003[END_REF]. Given that other viruses, such as Respiratory Syncytial Virus (RSV) and Influenza, mainly spread during the winter, the additional upsurge in RVGE exacerbates the problem of overcrowding in emergency departments and paediatric wards [START_REF] Roux | Nosocomial viral infections in a pediatric service: example of rotaviral gastroenteritis and respiratory syncytial viral bronchiolitis[END_REF] [START_REF] Thuret | Prospective follow-up of hospital-acquired diarrhoea in 28 paediatric wards of the south-east part of France during a winter season[END_REF].

The results of our study suggest that RV vaccination (90% coverage) could eliminate 45 hospitalizations per 10000 children aged less than 5 years. However, on basis of the literature, and of the results of our sensitivity analysis, we can affirm that RV vaccination could eliminate a maximum (100% coverage) of 64 hospitalizations per 10000 children aged less than 5 years.

In routine hospital practice, laboratory tests are not frequently used to confirm RV infection in children examined in the ED and/or kept in SO; for this reason, the proportion of diarrhoeal episodes due to RV cannot be directly determined. Testing for RV infection in faeces is only carried out in hospitalised children.

Our calculation of the number of avoidable cases confirmed that universal vaccination would reduce the impact of RV infection on ED admissions, hospitalisations and admissions to the SO unit; similar finding have been reported by other authors [START_REF] Huet | Burden of paediatric rotavirus gastroenteritis and potential benefits of a universal rotavirus vaccination programme with RotaTeq in France[END_REF] [START_REF] Panatto | Burden of rotavirus disease and cost-effectiveness of universal vaccination in the Province of Genoa (Northern Italy)[END_REF]. These results are confirmed by recent data on surveillance in the USA, where the introduction of RV vaccination into the paediatric immunization schedule has reduced the burden of RV gastroenteritis [START_REF]Delayed onset and diminished magnitude of rotavirus activity-United States, November 2007-May 2008[END_REF].

When evaluating the introduction of a new vaccination programme, it is necessary to consider the clinical, economic and social damage caused by the disease. RV illness has a considerable impact on society, as it mainly strikes children under 5 years of age, who are dependent on their families (e.g. productivity loss due to the absenteeism from work of one parent). RV disease does not only cause suffering for the infected child, but also anxiety and stress for the family. Furthermore, the universal vaccination of children <1 year would not entail a significant additional workload for healthcare services, as the vaccine can be administered simultaneously with other vaccines and vaccination can be inserted into the vaccination sessions scheduled for infants aged 3 and 5 months [START_REF]Word Health Organization (WHO)[END_REF].

Our study has the limitation that we did not collect the medical records of the RV-negative hospitalised children, which would have enabled us to assess the severity of their gastroenteritis and to compare the severity of RV disease with that of disease caused by other etiological agents of diarrhoea.

In conclusion, our study confirms that acute RV gastroenteritis is a major public health problem and that it has a heavy impact on Italian healthcare services and on society. This research adds information on the demographic and clinical features of RV disease in Italy and the results could be used to evaluate the change in the incidence of RV disease as a result of the introduction of vaccination.

On the basis of our estimate of the potentially avoidable cases of RV, it is reasonable to expect that a universal vaccination programme would substantially reduce the burden of RV on the healthcare system in Italy, particularly on hospitalisations. 1 A -Sensitivity analysis: number of cases avoided by RV vaccination on varying the number of RVpositive cases B -Sensitivity analysis: number of cases avoided on varying the efficacy of the RV vaccine C -Sensitivity analysis: number of cases avoided on varying RV vaccination coverage

Caption to Figure

  The 776 hospitalised children were tested for RV. In 27.83% (95% CI 24.67-30.98) (216 patients: 110 females and 106 males), the RV test proved positive. Therefore, the RV hospitalization rate was 55 per 10000 children aged less than 5 years in Genoa in 2006. Table2shows the distribution of RV-positive and -negative cases by age-group among the hospitalised children with community-acquired RV infection. We analyzed the medical records of RV-positive children with community-acquired infection and nosocomial infection; 22 cases (12 females and 10 males) were classified as nosocomial infections because the onset of gastrointestinal symptoms occurred 3 or more days after hospitalisation due to other causes.We also registered admission diagnoses in children hospitalised with community-acquired RV infection. Briefly,theadmission diagnosis was dehydration in 70 subjects (36.08%, 95% CI 29.32-42.83), vomiting in 36 (18.55%, The average age of children hospitalised with community-acquired RV infection was 27.79 months + 16.64 SD (24.1 median age), and the length of hospitalisation was 4.18 days + 3.8 SD. The average age of hospitalised children with nosocomial RV infection was 23.61 months + 17.17 SD (22.55 median age). The RV disease prolonged their hospital stay by an average of 4.4 days + 2.7 in comparison with the main diagnosis on hospital admission and the main diagnosis on discharge.

	-12.86). Of these, 276 children (11.52%, 95% CI
	10.24-12.79) were only examined and then sent home; 1286 (53.67%, 95% CI 51.67-55.66) were kept in SO; for
	834 children (34.80%, 95% CI 32.90-36.71) hospitalisation was requested. Of these, 776 (32.38%, 95% CI
	30.51-34.25) were hospitalised and 58 declined hospitalisation (2.42%, 95% CI 1.80-3.03).
	95% CI 13.08-24.01), nausea and vomiting in 2 (1.03%, 95% CI 0-2.45), diarrhoea in 33 (17.01%, 95% CI
	11.72-22.29), fever in 28 (14.43%, 95% CI 9.48-19.37), gastroenteritis in 82 (42.26%, 95% CI 35.31-49.21) and
	other (abdominal pain, rhinitis, cough) in 37 (19.07%, 95% CI 13.6-24.6); it should be borne in mind that an
	individual child may have more than one admission diagnosis.

Table 3

 3 reports the Vesikari scores and clinical characteristics of children with community-acquired versus nosocomial RV gastroenteritis. The cases were broken down according to severity by means of the method described by Vesikari

Table 1 :

 1 Parameters used in the analysis of the potentially avoided cases in the basic case and in the sensitivity

	analysis range
	Parameters	Base case	Sensitivity analysis	References
	Birth cohort size	39,176	-	ISTAT (12)
	Total number of admissions to	2,396	-	Data collected in the present
	Emergency Department for			study
	gastroenteritis
	Number of cases of gastroenteritis	276	-	Data collected in the present
	examined only in ED			study
	Number of cases of gastroenteritis in	1,286	-	Data collected in the present
	short observation			study
	% RV positive cases at Emergency	33.0	28.3-37.9	Forster J (Pediatrics, 2009)
	Department or in Short Observation
	Number of gastroenteritis cases	776	-	Data collected in the present
	hospitalised with community-acquired			study
	infection
	Number of RV-positive cases	216	-	Data collected in the present
	hospitalised with community-acquired			study
	infection
	Number of RV-positive cases	22	-	Data collected in the present
	hospitalised with nosocomial infection			study
	% RV-positive cases hospitalised	27.8	26.7-40.2	Data collected in the present
				study
				Forster J (Pediatrics, 2009)
	Vaccine efficacy (Rotarix®)	87.1	79.6-92.1%	Vesikari T (Lancet, 2007)
	any severity
	Vaccine efficacy (Rotarix®)	90.4%	85.1-94.1%	Vesikari T (Lancet, 2007)
	severe forms
	Coverage	90%	60%-100%	Assumption based on expert
				opinion

TABLE 3 :

 3 Vesikari score and comparison of clinical characteristics of community-acquired versus nosocomial

	RV gastroenteritis
	Signs and symptoms	Points	RVGE community	RVGE	OR	P value
		Vesikari	acquired	Nosocomial
		Score	(N°194)	(N°22)
	Duration of vomiting
	(days)
	0	0	64/216	3/22
	1	1	76/216	10/22
	2	2	38/216	7/22
	>3	3	38/216	2/22	1.3 (0.5-3.1)	0.6
	Vomiting: frequency/day
	0	0	64/216	3/22
	1	1	88/216	13/22
	2-4	2	42/216	6/22
	> 5	3	22/216	0/22	0.9 (0.3-2.4)	1
	Duration of diarrhoea
	(days)
	0	0	35/216	2/22
	1-4	1	121/216	10/22
	5	2	28/216	5/22
	> 6	3	32/216	5/22	2.2 (0.8-5.2)	0.1
	Diarrhoea: frequency/day
	0	0	35/216	2/22
	1-3	1	126/216	11/22
	4-5	2	32/216	5/22
	> 6	3	23/216	4/22	2.0 (0.8-0.5)	0.1
	Body temperature °C
	< 37.1	0	19/216	3/22
	37.1-38.4	1	123/216	19/22

Acknowledgments

The authors thank: Manuela Rescali for her support in data collection; Roberto Scali and Roberto Arrighi for their assistance in extracting epidemiological data; Emanuela Piccotti for data collection in the Emergency Department, and Paolo Ferrari, Anna Pellettieri, Antonella Formiga, Laura Fenu and Cinzia Gatti for their technical support.

Thanks go to GlaxoSmithKline, who partially supported this study by providing two educational grants.


Milana Lisunova
email: milana.lisunova@fu-berlin.de

Attila Hildmann

Benjamin Hatting

Vitaliy Datsyuk

Stephanie Reich

Nanofibres of CA/PAN with high amount of carbon nanotubes by core-shell electrospinning

Keywords: Carbon nanotubes, A: Polymers, A: Fibres, E: Electro-spinning, B: Thermal properties

We prepared polyacrylonitrile (PAN) and cellulose acetate (CA) based nanofibres with high amount of carbon nanotubes (CNTs) by core-shell electrospinning. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) were used to evaluate the morphology and structure of the electrospun nanofibres. Raman spectroscopy (Raman) and TEM indicate alignment of CNTs in the polymer fibres.

Core-shell electrospinning improved the distribution and uniformity of the fibres. The loading of carbon nanotubes showed better thermal stability.

Introduction

Electrospinnning of cellulose and derived materials is a well known and well developed process for the production of nanofibres. Such fibres are used as filters, drugdelivery vehicles, and scaffolds for tissue cultures [START_REF] Liu | Ultrafine fibrous cellulose membranes from electrospinning of cellulose acetate[END_REF][START_REF] Pankonien | Electrospinning of cellulose and carbon nanotubecellulose fibers for smart applications[END_REF]. Electrospun cellulose acetate (CA) nanofibres with conductive fillers such as carbon nanotubes (CNTs) [START_REF] Reich | Carbon nanotubes: basic concepts and physical properties[END_REF] are also interesting for electro-active paper sensor and actuator [START_REF] Liu | Ultrafine fibrous cellulose membranes from electrospinning of cellulose acetate[END_REF]. CA/CNTs -based composites are promising materials for sensors, electromagnetic shielding materials, and antistatic films [START_REF] Rubenstein | Bioassay Chamber for Angiogenesis with Perfused Explanted Arteries and Electrospun Scaffolding[END_REF], due to the unique electronic, thermal, optical, and mechanical properties of CNTs [START_REF] Reich | Carbon nanotubes: basic concepts and physical properties[END_REF]. Additionally, carbon nanotubes were considered for a wide range of applications in nanotechnology, because of their superior material properties such as low density and high aspect ratio [START_REF] Lisunova | Percolation behaviour of ultrahigh molecular weight polyethylene/multi-walled carbon nanotubes composites[END_REF]. Electrospinning CA solution with a filler fraction greater than 4 %wt. resulted in the formation of short and beaded fibres [START_REF] Rubenstein | Bioassay Chamber for Angiogenesis with Perfused Explanted Arteries and Electrospun Scaffolding[END_REF].

Elctrospinning CA with high concentration of CNTs was limited by viscosity [START_REF] Seoul | Electrospinning of Poly(vinylidene fluoride)/dimethylformamide solutions with carbon nanotubes[END_REF].

Incorporating a high fraction of filler in electrospun CA nanofibres was achieved by adding polyacrylonitrile (PAN) or polyethylene oxide (PEO) [START_REF] Chen | Electrospun cellulose acetate fibers containing chlorhexidine as a bactetricide[END_REF]. Such blending of polymers leads to the enhancement of the homogeneity and the stability of the electrospun jet, but the immiscibility of polymers is problematic [START_REF] Chen | Electrospun cellulose acetate fibers containing chlorhexidine as a bactetricide[END_REF]. Core-shell electrospinning seems to be promising for combining immiscible polymer solutions while keeping their individual properties [START_REF] Lallave | Filled and Hollow carbon nanofibers by coaxial electrospinning of alcell lignin without binder polymers[END_REF][START_REF] Han | Preparation and characterization of coreshell structured nanofibers by coaxial electrospinning[END_REF][START_REF] Sun | Compound coreshell polymer nanofibers by co-electrospinning[END_REF][START_REF] Kuo | Poly(3-hexylthiophene)/poly(methyl methacrylate) core-shell electrospun fibers for sensory applications[END_REF][START_REF] Li | Nanofibers of Conjugated polymers prepared by electrospinning with a two-capillary spinneret[END_REF].

In this work we report the production of core-shell nanofibres based on cellulose acetate and polyacrylonitrile filled with a large fraction of carbon nanotubes. We investigate and discuss the morphology, structure and thermal stability of the nanofibers.

Experimental

Materials

The CNTs used in this work were Multiwalled carbon nanotubes (MWCNTs) "Baytubes C150P" supplied by Bayer Materials Science AG. The MWCNTs were synthesized by the chemical vapour deposition (CVD). Statistical study of the TEM images (Figure 1) of CNT showed they are multiwalled nanotubes with an outer diameter d 20-40 nm and their length l n is about tens of microns (µm). The density of the MWCNT walls was estimated according to the ENI 8060 ρ f =1.2 -1.7 g/cm 3 .

Electrospinning setup

A schematic illustration of the experimental setup for electrospinning is presented in Fig. 2. Coaxial needles containing the core and shell precursor solutions were used to produce the fibres. 10 %wt. of PAN in DMF with 15 %wt. of CNTs was used to produce the PAN/CNTs fibres. 15w% of CA in DMAc/acetone (2 to 1) and 10 %wt. of PAN in DMF with 35 %wt. of CNTs were used to produce the core and shell of the CA/PAN-CNTs nanofibres, respectively. The fraction of nanotubes in both samples is given to the polymer matrix. The flow rates of both solutions were 0.2 ml/h. The tip of the core needle and the collector were connected to the high-voltage power supply. The tip-to-collector distance was 15-20 cm, and the electrical potential difference was 30 kV.

Fig. 2. Schematic illustration of the electrospinning setup

Characterization

The calorimetry experiments were performed using a differential-scanning calorimeter DSC-PT1 (Linseis Messgeräte GmbH), equipped with sub-ambient temperature operation. The heat flux and temperature were calibrated with a sapphire standard for the same scan rate as used in our experiments. The measurements were carried out in the temperature range of 350-678 K, in the heating mode. The samples were encapsulated into small-volume aluminum hermetic pans. In order to keep the melt peaks as narrow as possible, the masses of samples were only 2-3 mg.

The structure of MWCNTs and the MWCNTs/PAN nanofibres were examined using the TEM (JEM 100 C, Jeol, operating at accelerating voltage 100 kV). The surface morphology of the nanofibres was studied by atomic force microscopy (AFM, Park AFM, Schaefer Technologie GmbH). Raman spectroscopic analysis of MWCNTs/PAN nanofibres was carried out using the Xplora Raman spectrometer (Horiba Scientific), under ambient conditions. The 514.5 nm line from an Ar + laser was used for excitation at a power of 6 mW. Ultrasonication was conducted using a sonifier equipped with a cup horn Sonopuls HD3100 (Bandelin), which was operated at a frequency of 10 kHz with output power 100 W. The CNTs in polymer solution were sonicated for 30 min.

Results and discussions

Morphology

Figure 3 presents the already introduced TEM images of the PAN/CNTs (a, c) and CA/PAN-CNTs (b, d) composites. The fraction of nanotubes is the same in both samples (15 wt%); the fibres were prepared by electrospinning (Fig. 3 (a,c)) and coreshell electrospinning (Fig. 3 (b,d Investigation of the surface morphology by AFM (see Fig. 4) shows that core-shell electrospinning leads to the formation of more smooth fibres compared to the fibres produced by electrospinning in Fig. 4 (a). Moreover, it is clearly proved that the coreshell structure reduced the amount of beads and defects. The effect of core-shell electrospinning on the distribution and packing of the fibre was also reported by Han [START_REF] Han | Preparation and characterization of coreshell structured nanofibers by coaxial electrospinning[END_REF].

Well defined and uniform fibres were observed for the combination of Nylon-6 (core)polyurethane (shell) [START_REF] Han | Preparation and characterization of coreshell structured nanofibers by coaxial electrospinning[END_REF], poly(ethylene oxide) (shell) and polysulfone (core) [START_REF] Sun | Compound coreshell polymer nanofibers by co-electrospinning[END_REF], poly(methyl methacrylate) (core) and Poly(3-hexylthiophene) (shell) [START_REF] Kuo | Poly(3-hexylthiophene)/poly(methyl methacrylate) core-shell electrospun fibers for sensory applications[END_REF], poly ( where T' is the temperature at peak maximum in the DSC response curve, represents the heating rate of the sample and R is the universal gas constant. The activation energy of degradation ∆E a of PAN increases with CNTs loading (see Table 1). This result can be explained by the energy barrier numbers in the transport process; it is in agreement with previously reported data for sodium aluminum hydride and lithium aluminum hydride composites filled by CNTs. ∆E a of CA slightly increases, i.e., CNTs are not included into the morphological structure of CA. The improved ∆E a of PAN also confirms the deagglomeration and uniform distribution of CNTs [START_REF] Lebovka | Phase transitions, intermolecular interactions and electrical conductivity behavior in carbon multiwalled nanotubes/nematic liquid crystal composites[END_REF][START_REF] Gong | Surfactant-assisted processing of carbon nanotube/polymer composites[END_REF][START_REF] Cadek | Morphological and mechanical properties of carbon-nanotube-reinforced semicrystalline and amorphous polymer composites[END_REF][START_REF] Kim | Thermal and electrical properties of poly(l-lactide)-graft-multiwalled carbon nanotube composites[END_REF].

Conclusion

Core-shell electrospinning improves the homogeneous distribution and uniformity of fibres based on PAN and CA with a high amount of CNTs. DSC data reveal an increase of the degradation temperature for CA and PAN with CNTs loading. The improved thermal properties possibly reflect the deagglomeration and alignment of CNTs in the polymer fibres. The enhancement of activation energies of degradation of polymer fibres are believed to be due to the ordered structure of CNTs.

Figure captions

Fig. 1 .

 1 Fig. 1. Typical TEM image of multiwalled carbon nanotubes Cellulose acetate (CA, with density ρ in = 1.184 g/cm 3 , melting temperature T m =280 0 C) and polyacrylonitrile (PAN, with density ρ in = 1.184 g/cm 3 , melting temperature T m =317 0 C) were obtained from Aldrich. The N,N-dimethyl-formamide (DMF), dimethyl-acetamide (DMAc) and acetone (Ac) were purchased from Aldrich and used without any purification as solvents with boiling points T b =153 0 C, T b =164 0 C and T b =56 0 C, respectively.

  )). Examination of the images reveals that the MWCNTs are homogeneously distributed inside the polymeric fibres and aligned along the axes of the nanofibres. The alignment of CNTs by electrospinning was also investigated by Gogotsi et.al. [13-15]. According to their hypothesis alignment of CNTs in the nanofibre occurs through two mechanisms: flow-confinement-and charge-induced alignment during the elctrospinning process.

Fig. 3 .

 3 Fig. 3. TEM images of the PAN/CNTs (a, c) and CA/PAN-CNTs (b, d) nanofibres

  were about 40-50 nm PAN/CNTs and 150 nm for the CA/PAN-CNTs nanofibres. The thicker diameter of the CA/PAN-CNTs results from the structural organization with an inner diameter of CA (core) around 60 nm and outer layer of PAN-CNTs (shell) around 50 nm.

Fig. 4 .

 4 Fig. 4. AFM images of the PAN/CNTs (a) and CA/PAN-CNTs (b) nanofibres

Fig. 5 .Figure 6 Fig. 6 .

 566 Fig. 5. Raman spectra of CA/PAN-CNTs nanofibres for the polarization parallel ( )

Fig. 7 .

 7 Fig. 7. DSC curves of CA/PAN-CNTs nanofibres at 10, 15, 20 K/min heating rates.

Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .Fig. 6 .Fig. 7 .

 1234567 Fig. 1. Typical TEM image of multiwalled carbon nanotubes

Table 1 .

 1 The activation energies for electrospun PAN, CA, PAN/CNTs and CA/PAN-CNTs.

		E a (kJ/mol)
	PAN	E a , PAN 4.299
	PAN/CNTs	E a , PAN 4,361
	CA	E a , PAN 4,598
	CA/PAN-CNTs	E a , PAN 4,316 E a , CA 4,602

Acknowledgement

This work was supprovied by the BMBF Project Format CNTherm. The authors thank Solve Soren for the TEM investigation.


Maria Tärnberg
email: maria.tarnberg@liu.se

Åse Östholm Balkhed

Hans-Jürg Monstein

Anita Hällgren

Håkan Hanberger

Lennart E Nilsson

In vitro activity of beta-lactam antibiotics against CTX-M-producing Escherichia coli

Keywords: Extended-spectrum beta-lactamases, susceptibility, MIC

Objectives: Beta-lactam antibiotics have been discussed as options for treatment of infections caused by multiresistant ESBL-producing bacteria if the MIC is low. The objective of this study was to investigate the in vitro activity of different beta-lactam antibiotics against CTX-M-producing E. coli.

Methods: 198 isolates of E. coli, with ESBL-phenotype were studied. PCR-amplification of CTX-M genes and amplicon sequencing were performed. MICs for amoxicillin-clavulanic acid, aztreonam, cefepime, cefotaxime, ceftazidime, ceftibuten, ertapenem, imipenem, mecillinam, meropenem, piperacillin-tazobactam and temocillin were determined with Etest.

Susceptibility was defined according to breakpoints of the EUCAST. MIC 50 and MIC 90 were calculated.

Results: Isolates with CTX-M group 9 showed higher susceptibility to the beta-lactam antibiotics tested than isolates belonging to CTX-M group 1. More than 90 % of the isolates belonging to CTX-M group 9 were susceptible to amoxicillin-clavulanic acid, ceftazidime, ceftibuten, piperacillin-tazobactam and temocillin. The susceptibility was high to mecillinam, 91 %, regardless of CTX-M group. All isolates were susceptible to imipenem and meropenem and 99 % to ertapenem.

Conclusions:

This study shows significant differences in susceptibility to different betalactam antibiotics among the CTX-M-producing E. coli isolates and a significant difference for many antibiotics tested between the CTX-M-producing groups 1 and 9. The good in vitro activity of other beta-lactam antibiotics than carbapenems indicate that clinical studies are warranted to examine the potential role of these beta-lactam antibiotics in treatment of infections caused by multiresistant ESBL-producing E. coli.

Introduction

Until the early twenty-first century many reports of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae described nosocomial outbreaks due to SHV-producing Klebsiella pneumoniae, with TEM as the other major enzyme group frequently found in Enterobacteriaceae. Today, ESBL-producing Escherichia coli are an increasing problem worldwide, including low-prevalence areas such as Scandinavia [1][START_REF] Fang | Molecular epidemiology of extended-spectrum beta-lactamases among Escherichia coli isolates collected in a Swedish hospital and its associated health care facilities from 2001 to 2006[END_REF][START_REF] Kjerulf | The prevalence of ESBL-producing E. coli and Klebsiella strains in the Copenhagen area of Denmark[END_REF][START_REF] Lytsy | The first major extended-spectrum beta-lactamase outbreak in Scandinavia was caused by clonal spread of a multiresistant Klebsiella pneumoniae producing CTX-M-15[END_REF][START_REF] Naseer | Nosocomial outbreak of CTX-M-15-producing E. coli in Norway[END_REF][START_REF] Nyberg | Detection and molecular genetics of extended-spectrum betalactamases among cefuroxime-resistant Escherichia coli and Klebsiella spp. isolates from Finland, 2002-2004[END_REF], with CTX-M enzymes most commonly found.

There are now more than 90 different CTX-M type beta-lactamases described [7], and divided into five different clusters reflecting similarity on the amino-acid sequence level; CTX-M group 1, CTX-M group 2, CTX-M group 8, CTX-M group 9 and CTX-M group 25 [START_REF] Bonnet | Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes[END_REF].

When reporting antimicrobial susceptibility of invasive isolates to the European Antimicrobial Resistance Surveillance System (EARSS), ESBL-producing E. coli and Klebsiella spp. have been considered resistant to the whole beta-lactam group except carbapenems [START_REF]European Antimicrobial Resistance Surveillance System[END_REF]. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) previously stated in their Expert Rules that ESBL-producing isolates sensitive for cephalosporins should be regarded as intermediate, and intermediate isolates should be regarded as resistant [START_REF]Expert Rules in Antimicrobial Susceptibility Testing, The European Committee on Antimicrobial Susceptibility Testing (EUCAST)[END_REF], but as of the end of April 2010 changed their recommendations [START_REF]European Committe on Antimicrobial Susceptibility Testing[END_REF]. Since ESBL-producing E. coli have been considered non-susceptible to all beta-lactam antibiotics except carbapenems for a long time, some laboratories may still report these bacteria as cross-resistant to all penicillins and cephalosporins, without previous determination of minimum inhibitory concentration (MIC).

Recent studies have shown that inadequate empirical therapy (i.e. oxyimino-cephalosporins or other drugs tested in vitro as resistant) of serious infections is more often likely to occur in patients infected by ESBL-producing E. coli, and that mortality in this group is higher compared to controls infected by non-ESBL E. coli [START_REF] Melzer | Mortality following bacteraemic infection caused by extended spectrum beta-lactamase (ESBL) producing E. coli compared to non-ESBL producing E. coli[END_REF][START_REF] Schwaber | Mortality and delay in effective therapy associated with extended-spectrum beta-lactamase production in Enterobacteriaceae bacteraemia: a systematic review and meta-analysis[END_REF][START_REF] Cordery | Evaluation of risk factors for the acquisition of bloodstream infections with extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella species in the intensive care unit; antibiotic management and clinical outcome[END_REF][START_REF] Gudiol | Bacteraemia due to extended-spectrum {beta}-lactamase-producing Escherichia coli (ESBL-EC) in cancer patients: clinical features, risk factors, molecular epidemiology and outcome[END_REF][START_REF] Ortega | Analysis of 4758 Escherichia coli bacteraemia episodes: predictive factors for isolation of an antibiotic-resistant strain and their impact on the outcome[END_REF]. Several studies have also shown that the increasing mortality is due the inadequate empirical therapy [START_REF] Rodriguez-Bano | Bacteremia due to extended-spectrum beta -lactamaseproducing Escherichia coli in the CTX-M era: a new clinical challenge[END_REF][START_REF] Tumbarello | Predictors of mortality in patients with bloodstream infections caused by extended-spectrum-beta-lactamase-producing Enterobacteriaceae: importance of inadequate initial antimicrobial treatment[END_REF][START_REF] Tumbarello | Bloodstream infections caused by extended-spectrum-betalactamase-producing Escherichia coli: risk factors for inadequate initial antimicrobial therapy[END_REF][START_REF] Song | Clinical outcomes of spontaneous bacterial peritonitis due to extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella species: a retrospective matched case-control study[END_REF]. However, it has been discussed if ESBLs, regardless of MIC, always should be judged as cross resistant to all cephalosporins. Occasionally successful treatments with beta-lactam antibiotics other than carbapenems of infections caused by ESBL-producing bacteria have been reported [START_REF] Bin | Outcome of cephalosporin treatment of bacteremia due to CTX-M-type extended-spectrum beta-lactamase-producing Escherichia coli[END_REF][START_REF] Lagace-Wiens | Treatment of lower urinary tract infection caused by multidrug-resistant extended-spectrum-beta-lactamase-producing Escherichia coli with amoxicillin/clavulanate: case report and characterization of the isolate[END_REF][START_REF] Nicolle | Successful treatment of ctx-m ESBL producing Escherichia coli relapsing pyelonephritis with long term pivmecillinam[END_REF][START_REF] Rodriguez-Bano | Community infections caused by extended-spectrum betalactamase-producing Escherichia coli[END_REF][START_REF] Gupta | Clinical efficacy of temocillin[END_REF].

Thus it is of great value to thoroughly investigate ESBL-producing and in particular CTX-Mproducing, E. coli regarding activity of the beta-lactam antibiotics to increase the treatment options for these infections. In this study we determined the MICs of twelve different betalactam antibiotics in a population of ESBL-producing E. coli from a low-prevalence area.

Material & methods

Bacterial isolates

One hundred ninety eight isolates of E. coli, collected during January 2002 until December 2007 at the Clinical Microbiology Laboratory, Linköping University Hospital, Sweden, that possessed phenotypic ESBL-characteristics (as deduced by ESBL Etest with cefotaxime and ceftazidime with and without clavulanic acid, performed by the routine laboratory) were included in this study. Isolates from the same patient with identical antibiogram and sample source were excluded. The isolates were mainly of urinary tract origin (63 %). Wounds and hygiene screenings accounted for 15 % and 14 %, respectively, and blood for 6 %.

PCR-amplification and DNA-sequencing

PCR-amplifications of CTX-M genes were carried out using modified universal forward and reverse primers under conditions described earlier [START_REF] Monstein | Molecular identification of CTX-M and blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons[END_REF]. PCR-amplicons were then sequenced using the M13 sequence primer and edited and compared as described in detail previously [START_REF] Monstein | Molecular identification of CTX-M and blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons[END_REF], as to subgroup the isolates further into CTX-M group 1, 2, 8, 9 or 25.

MIC-determination

MIC-determinations for twelve different beta-lactam antibiotics were performed using Etest (BioMerieux, Marcy L'Etoile, France) according to the manufacturer's instructions. E. coli ATCC 25922 was used as a reference strain. The antibiotics tested were amoxicillinclavulanic acid, aztreonam, cefepime, cefotaxime, ceftazidime, ceftibuten, ertapenem, imipenem, mecillinam, meropenem, piperacillin-tazobactam and temocillin.

Breakpoints

Species related MIC breakpoints of the EUCAST were used to classify isolates as susceptible (S), intermediate or resistant (R) [27]. For temocillin two breakpoints according to the British Society for Antimicrobial Chemotherapy (BSAC) were used, 8 and 32 mg/L respectively [START_REF]BSAC methods for antimicrobial susceptibility testing[END_REF]. See table 1.

Statistics

Differences in antibiotic susceptibility between CTX-M groups 1 and 9 were analysed with non-parametric statistics, using the Mann-Whitney test and software SPSS v. 15.0. Isolates were grouped as susceptible vs. intermediate and resistant. Results were considered significantly different when p <0.05.

Results

PCR-amplification and DNA-sequence analysis

Of the 198 E. coli isolates with an ESBL-phenotype, 188 isolates (95%) carried CTX-M genes. One hundred thirty two isolates (67 %) belonged to group 1, 55 isolates (28 %) belonged to group 9, and one isolate belonged to group 2.

Susceptibility testing

Cephalosporins. For ceftibuten there was a highly significant difference in susceptibility between CTX-M group 1 and 9 (p <0.001), with 10 % susceptible isolates in group 1 compared to 95 % in group 9. For ceftazidime a similar difference was observed (p <0.001), 8 % susceptible isolates in group 1 compared to 95 % in group 9. For cefepime the overall susceptibility was low, 8 %, with a significant difference (p = 0.030) seen in CTX-M group 1 with 3 % susceptible isolates compared to 11 % in group 9. No isolates carrying CTX-M genes were susceptible to cefotaxime (table 1).

Penicillins. When applying the high BSAC breakpoint (32 mg/L) for temocillin all isolates were considered susceptible but the lower breakpoint (8 mg/L) decreased the susceptibility to 83 %. Using the lower BSAC breakpoint, the difference in temocillin susceptibility between CTX-M group 1 (79 %) and group 9 (91 %) was statistically significant (p = 0.048). Ninety one percent of all the isolates were susceptible for mecillinam (table 1).

Penicillin/beta-lactamase inhibitor combinations. For amoxicillin-clavulanic acid there was a significant difference in susceptibility between CTX-M group 1 and 9 (p = 0.014), with 80 % susceptible isolates in group 1 compared to 95 % susceptible in group 9 (table 1). Also for piperacillin-tazobactam there was a statistically significant difference in susceptibility between CTX-M group 1 and group 9 (p = 0.009), 82 % and 96 % susceptible isolates, respectively (table 1).

Aztreonam. For aztreonam there was a highly significant difference in susceptibility between CTX-M group 1 and 9 (p <0.001), with 0 % susceptible isolates in group 1 compared to 18 % in group 9 (table 1).

Carbapenems. All isolates were susceptible to imipenem and meropenem, 99 % were also susceptible to ertapenem (table 2).

Discussion

The main findings of this study are the substantial differences in susceptibility to different beta-lactam antibiotics among the studied CTX-M-producing E. coli isolates in general and the big difference in susceptibility between the CTX-M-producing groups 1 and 9 for several antibiotics, in particular.

To our knowledge this is the most extensive study of the in vitro activity of several different beta-lactam antibiotics against a large collection of CTX-M-producing E. coli. Although ESBL-producing isolates hydrolyzes penicillins and cephalosporins, this study shows that MIC-determination of different beta-lactam antibiotics can be of value in finding treatment options. Comparisons of results with other investigators are often difficult, mainly because of lack of MIC-distributions in the presentations, no presentation of breakpoints used and a different distribution of ESBL-producing enzymes or no specification of the enzymes.

The MIC-distribution for cefotaxime, ceftazidime and cefepime in the present study is similar to the results in a British study by Livermore et al. [START_REF] Livermore | Orthodox and unorthodox clavulanate combinations against extended-spectrum beta-lactamase producers[END_REF], and in a Belgian study by Rodriguez-Villalobos et al. [START_REF] Rodriguez-Villalobos | In vitro activity of temocillin against extended spectrum beta-lactamase-producing Escherichia coli[END_REF], but when applying the current EUCAST breakpoints on their MICdistributions the rate of susceptibility differs. For cefotaxime the overall susceptibility rate of the British study is similar to ours (1 %) but for the Belgian MIC-distributions the susceptibility is as high as 14 %. For ceftazidime the corresponding figures are 34 % susceptible isolates in our study compared to 12 % in the UK-study and only 4 % in the Belgian study. Finally for cefepime the overall susceptibility rate is much higher in both the UK and Belgian studies, 28 -34 % compared to only 8 % in our study. Some of these differences may be explained by differences in production of different groups of ESBLenzymes among the isolates. In this study 65 % of the CTX-M enzymes were group 1 and 28 % group 9 compared to 74% CTX-M group 1 and only 3 % group 9 in the UK-study and approximately 40 % CTX-M group 1 and 5 % group 9 in the Belgian study. The differences in susceptibility seen between CTX-M group 1 and 9 for ceftazidime (8 and 94 %, respectively) are in concordance with a recent Norwegian study [START_REF] Tofteland | Effects of phenotype and genotype on methods for detection of extended-spectrum-beta-lactamase-producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in Norway[END_REF].

The MIC-distribution of ceftibuten among the ESBL-producing E. coli in the present study was wide, with MIC-values ranging from 0.125 ->128 mg/L. This has also been shown in a

Taiwanese study [START_REF] Liao | In vitro activities of 16 antimicrobial agents against clinical isolates of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in two regional hospitals in Taiwan[END_REF]. We found a big difference between the CTX-M groups with 95 % susceptible isolates in the CTX-M group 9 and 10 % in CTX-M group 1.

The overall susceptibility for mecillinam in this study is remarkably high, 91 %. There is one case report on successful treatment of pyelonephritis caused by a CTX-M-producing E. coli with mecillinam [START_REF] Nicolle | Successful treatment of ctx-m ESBL producing Escherichia coli relapsing pyelonephritis with long term pivmecillinam[END_REF], but the value of treatment of UTIs caused by ESBL-producing organisms with mecillinam needs to be further evaluated. Thomas et al. advice against use of mecillinam in serious infections due to an inoculum effect seen in vitro [START_REF] Thomas | Activity of mecillinam against ESBL producers in vitro[END_REF].

Treatment of lower urinary tract infections caused by CTX-M-producing E. coli is often limited to nitrofurantoin and fosfomycin due to frequent co-resistance to other oral preparations such as quinolones, trimethoprim and trimethoprim-sulfamethoxazole. During pregnancy and childhood treatment with fluoroquinolones is not recommended. In this perspective the possibility to treat with an oral beta-lactam agent is an option and the results in this study indicate good in-vitro activity for mecillinam and for ceftibuten among isolates in CTX-M group 9.

Temocillin is a penicillin only commercially available in Belgium and the UK, which has been showing promising effect in vitro on ESBL-producing isolates [START_REF] Rodriguez-Villalobos | In vitro activity of temocillin against extended spectrum beta-lactamase-producing Escherichia coli[END_REF][START_REF] Livermore | Activity of temocillin against prevalent ESBL-and AmpCproducing Enterobacteriaceae from south-east England[END_REF]. Clinical studies on the efficacy of temocillin in treatment of infections caused by ESBL-producing isolates are scarce. However, there is a case series of severe sepsis caused by ESBL-producing isolates, showing promising results with temocillin [START_REF] Gupta | Clinical efficacy of temocillin[END_REF]. In our study the overall susceptibility for temocillin was high, 83 or 100 %, depending on the breakpoint (8 or 32 mg/L) used. These susceptibility results similar to Rodriguez-Villalobos et al. (81 and 99 %, respectively) [START_REF] Rodriguez-Villalobos | In vitro activity of temocillin against extended spectrum beta-lactamase-producing Escherichia coli[END_REF], but differs from Livermore et al. (64 and >99%, respectively) [START_REF] Livermore | Activity of temocillin against prevalent ESBL-and AmpCproducing Enterobacteriaceae from south-east England[END_REF].

The overall susceptibility for amoxicillin-clavulanic acid was the same as for piperacillintazobactam (84%), and as high as 94 % for amoxicillin-clavulanic acid and 96 % for piperacillin-tazobactam for the strains belonging to CTX-M group 9.

The amoxicillin-clavulanic acid results in our study are similar to those from Sorlozano et al. [START_REF] Sorlozano | Activity in vitro of twelve antibiotics against clinical isolates of extended-spectrum beta-lactamase producing Escherichia coli[END_REF], but differs from others [START_REF] Hoban | In vitro activity of tigecycline against 6792 Gram-negative and Gram-positive clinical isolates from the global Tigecycline Evaluation and Surveillance Trial (TEST Program, 2004)[END_REF][START_REF] Oteo | Spread of Escherichia coli strains with high-level cefotaxime and ceftazidime resistance between the community, long-term care facilities, and hospital institutions[END_REF][START_REF] Prakash | Oral and parenteral therapeutic options for outpatient urinary infections caused by enterobacteriaceae producing CTX-M extended-spectrum betalactamases[END_REF]. Amoxicillin-clavulanic acid has been used for successful treatment of cystitis caused by multidrug resistant CTX-M 15 producing E. coli [START_REF] Lagace-Wiens | Treatment of lower urinary tract infection caused by multidrug-resistant extended-spectrum-beta-lactamase-producing Escherichia coli with amoxicillin/clavulanate: case report and characterization of the isolate[END_REF], but failed in 1 of 11 bloodstream infections [START_REF] Rodriguez-Bano | Bacteremia due to extended-spectrum beta -lactamaseproducing Escherichia coli in the CTX-M era: a new clinical challenge[END_REF].

In the study by Rodriguez-Villalobos et al. [START_REF] Rodriguez-Villalobos | In vitro activity of temocillin against extended spectrum beta-lactamase-producing Escherichia coli[END_REF], 82 % of the ESBL-producing E. coli were susceptible to piperacillin-tazobactam applying the EUCAST breakpoints. Using CLSI breakpoints (susceptible ≤16 mg/L), several studies show moderate to high activity (69-95 %) for piperacillin-tazobactam [START_REF] Sorlozano | Activity in vitro of twelve antibiotics against clinical isolates of extended-spectrum beta-lactamase producing Escherichia coli[END_REF][START_REF] Hoban | In vitro activity of tigecycline against 6792 Gram-negative and Gram-positive clinical isolates from the global Tigecycline Evaluation and Surveillance Trial (TEST Program, 2004)[END_REF][START_REF] Colodner | First national surveillance of susceptibility of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. to antimicrobials in Israel[END_REF][START_REF] Sader | Comparative activities of cefepime and piperacillin/tazobactam tested against a global collection of Escherichia coli and Klebsiella spp. with an ESBL phenotype[END_REF]. Successful treatment with piperacillin-tazobactam of infections caused by ESBL-producing bacteria has also been reported [START_REF] Gavin | Clinical correlation of the CLSI susceptibility breakpoint for piperacillin-tazobactam against extended-spectrum-beta-lactamase-producing Escherichia coli and Klebsiella species[END_REF]. According to Peterson even severe infections should be considered treatable when the MIC value imply so [START_REF] Peterson | Antibiotic policy and prescribing strategies for therapy of extendedspectrum beta-lactamase-producing Enterobacteriaceae: the role of piperacillintazobactam[END_REF], but most authors advise against the use of piperacillin-tazobactam in ESBL infections.

The overall susceptibility rate for aztreonam was extremely low (8 %) and for strains belonging to CTX-M group 1 none of the strains were susceptible. The differences seen between CTX-M group 1 and 9 for aztreonam are in concordance with a recent Norwegian study [START_REF] Tofteland | Effects of phenotype and genotype on methods for detection of extended-spectrum-beta-lactamase-producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in Norway[END_REF].

The susceptibility for carbapenems was very high (99 -100 %) and in agreement with worldwide studies [START_REF] Rodriguez-Villalobos | In vitro activity of temocillin against extended spectrum beta-lactamase-producing Escherichia coli[END_REF][START_REF] Liao | In vitro activities of 16 antimicrobial agents against clinical isolates of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in two regional hospitals in Taiwan[END_REF][START_REF] Sorlozano | Activity in vitro of twelve antibiotics against clinical isolates of extended-spectrum beta-lactamase producing Escherichia coli[END_REF][START_REF] Colodner | First national surveillance of susceptibility of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. to antimicrobials in Israel[END_REF][START_REF] Hernandez | Comparative in vitro activity of ertapenem against extendedspectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolated in Spain[END_REF][START_REF] Kiremitci | In vitro activity of ertapenem and other carbapenems against extended-spectrum beta-lactamase producing Escherichia coli and Klebsiella pneumoniae clinical isolates in a tertiary care center in Turkey[END_REF]. All isolates were susceptible to imipenem and meropenem and only two isolates were resistant to ertapenem. Carbapenems are still a recommended therapy against invasive infections caused by ESBL-producing bacteria.

In conclusion this study shows significant difference in susceptibility to different beta-lactam antibiotics among CTX-M-producing E. coli. Isolates with ESBL enzymes belonging to CTX-M group 9 were in general more susceptible to these antibiotics than group 1. Further comparative studies using different methods for MIC determination (such as Etest, agar and broth dilutions) are needed to confirm the good in vitro activity of beta-lactam antibiotics other than carbapenems, against CTX-M-producing E. coli, as demonstrated in this study.

Studies of different isolates with MICs close to the breakpoints with time killing curve experiments and animal in vivo experiments are also needed. However, most warranted are clinical studies to examine the potential role of these beta-lactam antibiotics in the treatment of infections caused by multiresistant ESBL-producing E. coli.

Table 1 .

 1 MIC-distributions. Breakpoints according to a) BSAC and b) EUCAST (S≤/R>). MIC 50 in bold underlined, MIC 90 in bold italics.399

	Number of isolates with indicated MIC (mg/L)
	<0.064	0.064	0.125	0.25	0.5 1 2 4	8	16	32	64	128	>128	%S	%R	breakpoints
	Temocillin														8/8 a
	All E. coli, n = 198					1 15 67	82	30	3				83.3 16.7
	CTX-M group 1, n = 132					1 6 26	71	25	3				78.8 21.2
	CTX-M group 9, n = 55					6 34	10	5					91	9
	Mecillinam														8/8 b
	All E. coli		8	22	32 67 33 12	7	3	4			10	91.4	8.6
	CTX-M gr 1		3	14	25 45 23 9	6		2			5	94.7	5.3
	CTX-M gr 9		5	8	6	19 8 2		2				5	87.3 12.7
	Ceftibuten														1/1 b
	All E. coli		2	8	27 37 14 35	36	24	6	5	1	3	37.4 62.6
	CTX-M gr 1				4	9 12 35	33	24	6	5	1	3	9.8	90.2
	CTX-M gr 9		1	5	19 27 1	2						94.5	5.5
	Cefotaxime														1/2 b
	All E. coli	2				3 3	25	26	31	33	22	53	2.5	96.0
	CTX-M gr 1						4	7	18	33	20	50	0	100
	CTX-M gr 9					1	20	17	13		1	3	0	98.2
	Ceftazidime														1/8 b
	All E. coli		4	13	28 22 10 39	40	24	8	4	6		33.8 21.2
	CTX-M gr 1				2	9 8 32	40	24	7	4	6		8.3	31.1
	CTX-M gr 9		3	12	25 12 1 2							94.5	0
	Cefepime														1/8 b
	All E. coli		1	4	2	9 32 60	42	22	13	3	2	8	8.1	24.2
	CTX-M gr 1				2	2 7 38	37	22	13	2	2	7	3.0	34.8
	CTX-M gr 9		1			5 24 20	3			1		1	10.9	3.6
	Piperacillin-tazobactam														8/16 b
	All E. coli		2	2	5	30 72 30	26	11	5		5	10	84.3 10.1
	CTX-M gr 1			2	3	13 39 26	25	11	5		4	4	81.8	9.8
	CTX-M gr 9		2		2	15 29 4	1					2	96.4	3.6
	Amoxicillin-clavulanic acid														-/8 b
	All E. coli					47	120	31						15.7
	CTX-M gr 1					19	87	26						19.7
	CTX-M gr 9					23	29	3						5.5
	Aztreonam														1/8 b
	All E. coli		2	3	1	9 28 29	13	46	30	16	9	12	7.6	57.1
	CTX-M gr 1					4 7	12	43	29	16	9	12	0	82.6

Table 2 .

 2 MIC-distributions. Breakpoints according to EUCAST (S≤/R>). MIC 50 in bold underlined, MIC 90 in bold italics.401

		Number of isolates with indicated MIC (mg/L)
		<0.008 0.008 0.016 0.032 0.064 0.125 0.25 0.5 1	2	4	8	16	>16	%S	%R	breakpoints
	Imipenem											2/8
	All E. coli n = 198					28	161 8	1			100	0
	CTX-M group 1 n = 132					20	106 6				100	0
	CTX-M group 9 n = 55					5	47	2	1			100	0
	Meropenem											2/8
	All E. coli	1	33	127	29	6	2					100	0
	CTX-M gr 1		11	87	28	6						100	0
	CTX-M gr 9	1	16	35	1		2					100	0
	Ertapenem											0.5/1
	All E. coli	10	52	52	46	16	8	11 1	2		98.5	1.0
	CTX-M gr 1	4	21	34	38	15	8	11 1			99.2	0
	CTX-M gr 9	3	30	15	5					2		96.4	3.6

Acknowledgement

This study was presented as a poster at the 19 th European Congress of Clinical Microbiology and Infectious Diseases in Helsinki, Finland, 16-19 May 2009.

Funding

This work was supported by the Medical Research Council of Southeast Sweden.

Transparency declaration

Nothing to declare.

Authors' contributions

MT, ÅÖB, HJM, AH, HH and LEN participated in the conception, design, drafting of the manuscript, and final approval of the version to be published. MT was responsible for the laboratory work, analysis, statistical calculations and interpretations. MT, ÅÖB and LEN were responsible for the clinical strain collection.