Lutsar

Ü Parm

T Metsvaht

E Sepp

M.-L Ilmoja

M Pisarev

I Pauskar

M Pauskar

I Lutsar
email: irja.lutsar@ut.ee

H Pisarev

Impact of empiric antibiotic regimen on bowel colonization in neonates with suspected early onset sepsis

Impact of empiric antibiotic regimen on bowel colonization in neonates with suspected early onset sepsis

Introduction

Empiric use of antibiotics in neonatal intensive care units is common but their widespread use is not problem-free [START_REF] Donskey | The role of the intestinal tract as a reservoir and source for transmission of nosocomial pathogens[END_REF]. The issue that administration of antimicrobial agents causes disturbances in the ecological balance between the host and the microorganisms and thus has a potential to interfere with initial gut colonization in neonates cannot be ignored [START_REF] Sullivan | Effect of antimicrobial agents on the ecological balance of human microflora[END_REF].

A combination of gentamicin with a beta-lactam antibiotic such as penicillin G or ampicillin is the most recommended treatment for early onset sepsis (EOS) [START_REF] Young | Neofax: a manual of drugs used in neonatal care[END_REF][START_REF] Mehta | BNF49: British National Formulary[END_REF]. Studies have shown that broad spectrum antibiotics including ampicillin have led to increased rates of colonization by potentially pathogenic members of Enterobacteriaceae and Candida spp. [START_REF] Bonnemaison | Comparison of fecal flora following administration of two antibiotic protocols for suspected maternofetal infection[END_REF][START_REF] Stoll | Very low birth weight preterm infants with early onset neonatal sepsis: the predominance of gram-negative infections continues in the National Institute of Child Health and Human Development Neonatal Research Network, 2002-2003[END_REF][START_REF] Gewolb | Stool microflora in extremely low birthweight infants[END_REF]. On the other hand narrow spectrum penicillins like penicillin G have the least potential of interfering with normal gut colonization [START_REF] Bennet | Suppression of aerobic and anaerobic faecal flora in newborns receiving parenteral gentamicin and ampicillin[END_REF]. Still the number of comparative studies on this issue is limited. De Man et al. [START_REF] De Man | An antibiotic policy to prevent emergence of resistant bacilli[END_REF] compared the effect of amoxicillin plus cefotaxime with penicillin plus tobramycin on gut colonization with special attention to the emergence of resistant Enterobacteriaceae in two NICUs over a period of six months and showed that with the first regimen 18.8% of neonates became colonized with bacteria resistant to initial antibiotics compared to only 1.3% with the second regimen. Still, the study did not clarify whether the differences between the two antibiotic regimens were triggered by amoxicillin or cefotaxime or both. The use of ampicillin in intrapartum prophylaxis of group B streptococcal infection has been associated with the emergence of Escherichia coli as a major causative pathogen of EOS and probably an increase in ampicillin-resistance [START_REF] Stoll | Very low birth weight preterm infants with early onset neonatal sepsis: the predominance of gram-negative infections continues in the National Institute of Child Health and Human Development Neonatal Research Network, 2002-2003[END_REF][START_REF] Laugel | Effects of antenatal antibiotics on the incidence and bacteriological profile of early-onset neonatal sepsis. A retrospective study over five years[END_REF], but the data are not conclusive [START_REF] Jaureguy | Effects of intrapartum penicillin prophylaxis on intestinal bacterial colonization in infants[END_REF].

Based on the above, we assumed that ampicillin as an antibiotic with wider Gram-negative coverage interferes more with initial gut colonization than penicillin and has the potential of inducing emergence of ampicillin-resistant Gram-negative bacteria.

We chose gut colonization studies to compare the two antibiotic regimens because in neonates opportunistic bacteria of the gastrointestinal tract serve as the predominant source of subsequent bloodstream infections [START_REF] Donskey | The role of the intestinal tract as a reservoir and source for transmission of nosocomial pathogens[END_REF][START_REF] Westerbeek | The intestinal bacterial colonisation in preterm infants: a review of the literature[END_REF][START_REF] Graham Pl 3rd | The gastrointestinal tract serves as the reservoir for Gram-negative pathogens in very low birth weight infants[END_REF][START_REF] Costa | Mucosa or skin as source of coagulase-negative staphylococcal bacteraemia?[END_REF]. Systematic investigations in the field may have a role in understanding the changing spectrum of bacteria involved in late onset sepsis (LOS) and possibly other longterm outcomes [START_REF] Westerbeek | The intestinal bacterial colonisation in preterm infants: a review of the literature[END_REF].

In a cluster-randomized switch-over study in neonates at risk of EOS and requiring third level intensive care, the influence of ampicillin or penicillin, both combined with gentamicin, on gut colonization by aerobic, facultative anaerobic and ampicillin resistant bacteria was compared in an analysis taking into account other factors interfering with gut colonization.

Methods

Bowel colonization assessments were seeded in a study comparing the clinical efficacy of ampicillin and gentamicin to that of penicillin and gentamicin in risk factor based empiric treatment of EOS [START_REF] Metsvaht | Comparison of ampicillin plus gentamicin vs. penicillin plus gentamicin in empiric treatment of neonates at risk of early onset sepsis[END_REF].

Study setting

The study was conducted in two Estonian third level pediatric intensive care units (PICU) from August 2, 2006 until November 30, 2007. Both units admit patients up to 16 years; about 60-65% of them are neonates cared for in a separate area. The units are divided into four and five rooms, respectively; the number of neonates in a room varies from three to six. The nursing staff/infant ratio in the units is 1:2, but can be 1:3 occasionally. Gloves, gowns, caps and masks are used routinely in all aseptic procedures. Both units follow similar hospital infection prevention guidelines and strict antibiotic policy, in which narrow spectrum antibiotics and short courses are preferred. Patients colonised with alert microorganisms are isolated in separate rooms and cared for by separate nurses. Both units practice early intro-duction of enteral feeding with preference given to breast milk. Formula if needed is prepared centrally; donor milk is not used.

Study population

The study enrolled neonates aged less than 72 h, needing empiric therapy for proven or suspected EOS with penicillin G (25,000 IU/kg 8-12 hourly) or ampicillin (25 mg/kg 8-12 hourly) plus gentamicin (4-5 mg/kg 24-48 hourly, according to gestational age) according to the criteria described by Schrag et al. [START_REF] Schrag | Prevention of perinatal group B streptococcal disease. Revised guidelines from CDC[END_REF]. Patients who had received a different antibiotic regimen for more than 24 h, had suspicion of meningitis, necrotizing enterocolitis, peritonitis or severe sepsis with a history of isolation of microorganisms resistant to the study regimen from maternal urinary tract or birth canal or had other situations where the treating physician considered a different antibiotic regimen necessary were excluded.

Study design

During the first study period (from August 03, 2006 to March, 20 2007) in unit A ampicillin and in unit B penicillin G was used. After enrolling half of the patients required for proving clinical equivalence of the two antibiotic regimens the penicillins were switched so that during the second period (from March 21, 2007 to November 30, 2007) in unit A penicillin G and in unit B ampicillin was used (Fig. 1). If no clinical or laboratory signs of invasive infection developed and initial blood cultures remained negative, antibiotics were stopped on day 3. In case of clinical or culture proven infection initial antibiotic regimen could be continued if susceptible pathogens were involved or changed to a prespecified regimen depending on the antibacterial susceptibility of the isolate. For the empiric therapy of LOS cefuroxime, cefotaxime, ampicillin/sulbactam, or piperacillin/tazobactam alone or in combination with gentamicin were recommended. In case of severe sepsis or septic shock cefotaxime with gentamicin or meropenem with or without vancomycin was to be used. In ELBW neonates with birth weight below 800 g and vascular catheter(s) in place, addition of vancomycin was recommended until culture results became available.

Data collection

Basic demographic and clinical data are shown in Table 1. Feeding regimen was documented on days 1, 3 and 7 with patients categorized into the following groups based on the route of nutrition and the character of enteral feeds: (1) total parenteral nutrition (TPN) when enteral calories constituted less than 10% of total daily calories, (2) breastfeeding when breast milk constituted more than 10% of enteral feeds, and (3) formula feeding when formula constituted more than 89% of enteral feeds. Additional parenteral nutrition supplying up to 89% of daily caloric intake was accepted in the two latter groups.

Sampling

Monitoring of gut colonization had been implemented by the infection control services prior to the study with rectal samples collected with transport swabs (Nuova Aptaca, Canelli, Italy) on admission and twice a week thereafter until discharge from PICU or until day 60 whichever occurred first.

Laboratory methods

Transport swabs were stored at -20°C for a maximum of a week and processed in batches. After thawing the swabs were directly plated onto blood agar, MacConkey agar, MacConkey agar with 16 μg/ml of ampicillin and Saboraud agar. The blood and MacConkey agar plates were incubated at 37°C for 24-48 h in ambient air and Saboraud agar plates at 25°C for at least one week. Each morphologically different colony type was Gram stained and identified on species and genus level according to the Clinical and Laboratory Standards Institute criteria [START_REF] Clsi | Performance standards for antimicrobial susceptibility testing; fifteenth informational supplement[END_REF]. For final identification of enterobacteria and staphylococci API commercial kits (API 20E and API STAF; Biomérieux, and Marcy l'Etoile, France, respectively) and for identification of yeasts CHROMagar™Candida (BD BBL, Heidelberg, Germany) were employed. The Staphylococcus aureus strains were further tested for methicillin-resistance (MRSA) by determining nuc and mecA genes as described elsewhere [START_REF] Brakstad | Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene[END_REF][START_REF] Chambers | Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications[END_REF].

In this study any Gram-negative microorganism that grew on MacConkey agar with 16 μg/ml of ampicillin was termed ampicillin-resistant (AR).

Statistical analysis

The two treatment regimens were compared to each other with regards to number of patients colonized and colonization duration (CD); the latter describing the ratio of colonizing days per 100 PICU days. The number of colonizing days was counted from the first until the last positive culture and an extra two days were added to compensate for the sampling interval, which was three to four days.

The software programs Sigma Stat for Windows 2.0 (Jandel Corporation, USA) and R 2.6.2 (A Language and Environment, http://www.r-project.org) were used for statistical analysis. Hierarchical mixed effect models corrected for the study centre and treatment period were used in all comparisons. Differences in proportions were compared using Chi-square test. To compensate for the influence of other

Results

Study population and setting

A total of 465 neonates (age 0-28 days) were admitted to both units with 43% and 34% excluded in units A and B, respectively (Fig. 1). Exclusion for no need of early empiric antibiotic treatment was more common in unit A than in unit B (odds ratio [OR] 3.78; 95% CI 2.18-6.53), likely reflecting a difference in admitted population. A total of 283 patients were included, colonisation data were available in 276 (97.5%) of them, thus 139 neonates in the ampicillin and 137 in the penicillin group constituted the study population (Fig. 1).

As shown in Table 1 the population characteristics in both treatment groups were well balanced. The median duration of primary antibiotic regimen was 64 h (interquartile range [IQR] 57-138) in the ampicillin and 72 h (IQR 55-134) in the penicillin arm. The use of additional broad spectrum antibiotic classes in both regimens was similar (Table 2) with the median duration of 220 h (IQR 95-347) in the penicillin and 268 h (IQR 108-446) in the ampicillin group. The median number of rectal samples collected per patient was three; with IQR 2-5 in the ampicillin and 2-6 in the penicillin group. The aetiology of EOS and LOS is presented in Table 3. There was a greater number of LOS cases caused by S. epidermidis in the During the study two outbreaks of bloodstream infections (BSI) confirmed by pulse field gel electrophoresis (PFGE) were observed. In unit B an outbreak of MRSA infection involving three patients occurred during the penicillin treatment period and in unit A five patients had a BSI caused by K. pneumoniae (all strains intermediately resistant to ampicillin) during the ampicillin treatment period. The latter outbreak was likely responsible for the differences in K. pneumoniae colonization between units and treatment periods.

Influence of participating units on gut colonization

In unit B more patients were colonized with E. coli (OR= 2.31; 95% CI 1. [START_REF] Metsvaht | Comparison of ampicillin plus gentamicin vs. penicillin plus gentamicin in empiric treatment of neonates at risk of early onset sepsis[END_REF] respective AR Gram negative organisms followed a similar pattern, except for E. coli, in which AR strains occurred with similar frequency in both units.

Influence of treatment period on gut colonization

The gut colonization pattern in both treatment periods was similar except for K. pneumoniae (OR=2.25; 95% CI 1.17-4.34), S. haemolyticus (OR=1.92; 95% CI 1.10-3.36), and S. hominis (OR=6.46; 95% CI 2.16-19.28), all being more frequent colonizers during the first compared to the second period.

Influence of empiric antibiotic regimen on bowel colonization over time

As presented in Fig. 2a andb Gram-positive microorganisms were the first and most common colonizers of the rectal mucosa, followed by Gram-negative and Gramnegative AR bacteria and yeasts. Both antibiotic regimens had similar effects on early gut colonization with Gram negative microorganisms and Candida spp except for greater number of patients colonized by Acinetobacter spp on days 13-16, including AR Acinetobacter spp between day 10 and 16 in the penicillin compared with the ampicillin arm (Fig. 2b). As for the colonization by Gram positive organisms the following differences between the two antibiotics were observed: penicillin treatment resulted in significantly greater number of patients colonized with Enterococcus spp at all timepoints from day 6 to 16 and by S. aureus between days 3 and 5 than ampicillin (Fig. 2a).

Influence of empiric treatment regimen on gut colonization-model corrected for participating unit and treatment period

As both the treatment period and the participating unit affected bowel colonization, all further analyses were corrected for these two co-variates. First, the penicillin and ampicillin treatments were compared based on the number of colonized patients. With the exception of AR Acinetobacter spp found only in the penicillin arm (0 vs 8; p=0.008), the number of patients colonized with all other Gram negative organisms was similar in both treatment arms (Table 4). Again differences were observed among Gram-positive microorganisms. Compared with penicillin, ampicillin treatment was associated with twofold greater odds of colonization by S. haemolyticus (OR 2.22; 95% CI 1.2-4.12) and sixfold that of S. hominis (OR 6.46; 95% CI 2.12-19.67), whereas the odds of colonisation by Enterococcus spp and S. aureus were about one half (OR 0.5; 95% CI 0.3-0.83) and one third (OR 0.34; 95% CI 0.13-0.91), respectively (Table 4).

Second, we compared the CDs (days colonised per 100 ICU days) in the two treatment arms. In this respect penicillin and ampicillin treatments were more distinct than in the per subject analysis. Higher mean CD of K. pneumoniae (difference +7.8; p =0.004) was seen in the ampicillin arm and AR Serratia spp (-3.4; p = 0.011) and AR Acinetobacter spp (-2.3; p = 0.004) in the penicillin arm (Table 4). Among Gram positive bacteria the CDs followed similar trends as in the per subject analysis; ampicillin was associated with higher mean The following significant differences were observed: the rate of patients colonised with Enterococcus spp was greater in the penicillin as compared with ampicillin arm on days 6-9 (OR=2.99; CI 95% 1.23-7.25) and days 13-16 (OR=3.33; CI 95% 1. 29-8.63). The rate of patients colonised with S. aureus on days 3 to 5 was greater in the penicillin than in the ampicillin arm (p=0.012). b. Colonization with Gram-negative and Gram-negative AR microorganisms. Continuous lines present data of the penicillin and dotted lines of the ampicillin group. Enterobacteriaceae are represented by squares, AR Enterobacteriaceae by triangles, Acinetobacter spp by circles, and AR Acinetobacter spp by diamonds. The following significant differences were observed: the rate of patients colonized by Acinetobacter spp in the penicillin as compared with the ampicillin arm on days 13-16 (6 vs 0; p=0.009) and AR Acinetobacter spp on days 10-12 (6 vs 0; p=0.045) and days 13-16 (4 vs 0; p=0.046) CDs of S. haemolyticus (difference +12.2; p = 0.001) and S. hominis (+5.8; p = 0.001) but lower CDs of S. epidermidis (-8.5; p = 0.039) and Enterococcus spp. (-22.9; p = 0.001). Although colonization by S. aureus in per patients analysis was different, in CD analysis this did not reach significance at the p value of <0.05 (p = 0.054) (Table 4).

Influence of antibiotic regimen on bowel colonization in multivariate, risk factor adjusted mixed effect model analysis

Next we conducted a multivariate analysis including microorganisms that in the model corrected for the study centre and treatment period which reached significance at a p value of ≤0.1 (Table 4). In per subject analysis the differences that remained significant were the greater number of patients colonized with S. aureus (p=0.006) and Enterococcus spp (p=0.0009) in the penicillin arm and those with S. haemolyticus (p=0.039) and S. hominis (p= 0.009) in the ampicillin arm (Table 5).

>Similarly to univariate analysis described above, early empiric AB regimen remained an independent risk factor for the CD of all major Gram negative microorganisms (K. pneumonia, AR Serratia spp, and AR Acinetobacter spp) also in multivariate models (Table 5). Among Gram positive organisms ampicillin was still independently associated with higher CD of S. haemolyticus (p=0.001) and S. hominis (p=0.001) and penicillin with Enterococcus spp (p=0.001) and S. aureus (p=0.052), but the association between penicillin treatment and higher CD of S. epidermidis was of borderline significance (p=0.0725). In addition, Significant differences are presented in bold a Presents p value as odds ratio cannot be calculated ampicillin treatment, not reaching significance for the CD of Candida spp in univariate analysis (p=0.077) (Table 4), now became associated with higher CD of Candida spp (p=0.02) (Table 5).

Discussion

To our best knowledge this is the first prospective study comparing the effect of two widely used antibiotic regimenspenicillin and ampicillin, both combined with gentamicin-on initial mucosal colonization in neonates with suspected EOS, looking specifically at colonization with Gram-negative AR bacteria. We found that the main differences between these two regimens did not involve Enterobacteraceae but appeared in colonization with Gram positive microorganisms. In patients receiving penicillin containing regimen, colonization with S. aureus and Enterococcus spp occurred with greater frequency and that of S. haemolyticus and S. hominis with lower frequency than in those receiving ampicillin. As for Gram negative microorganisms and Candida spp, the number of colonized patients in both treatment arms was similar but greater CDs of K. pneumonia, AR Serratia spp and Candida spp were seen in the ampicillin arm and that of AR Acinetobacter spp in the penicillin arm.

Studies have shown that gut colonization of a neonate is a complex process influenced by several intrinsic and extrinsic factors [START_REF] Stoll | Very low birth weight preterm infants with early onset neonatal sepsis: the predominance of gram-negative infections continues in the National Institute of Child Health and Human Development Neonatal Research Network, 2002-2003[END_REF][START_REF] Gewolb | Stool microflora in extremely low birthweight infants[END_REF][START_REF] Jaureguy | Effects of intrapartum penicillin prophylaxis on intestinal bacterial colonization in infants[END_REF][START_REF] Westerbeek | The intestinal bacterial colonisation in preterm infants: a review of the literature[END_REF][START_REF] Tapiainen | Dynamics of gut colonization and source of intestinal flora in healthy newborn infants[END_REF][START_REF] Mackie | Developmental microbial ecology of the neonatal gastrointestinal tract[END_REF][START_REF] Edwards | Intrapartum antibiotic prophylaxis 1: relative effects of recommended antibiotics on gram-negative pathogens[END_REF][START_REF] Duman | Beta-lactam antibiotic resistance in aerobic commensal fecal flora of newborns[END_REF]. Thus, when evaluating the interference of early antibacterial therapy with this process the methodological considerations are of utmost importance. A major advantage of this study was the switch-over design that enabled accounting for the differences between the participating units and treatment periods. As shown here and by others [START_REF] Mammina | Surveillance of multidrug-resistant gram-negative bacilli in a neonatal intensive care unit: prominent role of cross transmission[END_REF], such differences leading to potential cross-colonization are unavoidable despite both units having fairly similar staffing, following similar treatment guidelines and strict infection control measures. Another strength was the statistical analysis conducted by us. First, to minimize the role of the participating unit and treatment period in the assessment of the antibiotic regimen, all comparisons were corrected for these two variables. Second, not only the number of colonized patients but also the time of colonization and its duration was considered, the latter potentially affecting cross-colonization more than the former [START_REF] Mammina | Surveillance of multidrug-resistant gram-negative bacilli in a neonatal intensive care unit: prominent role of cross transmission[END_REF]. Finally, also maternal, neonatal and environmental factors known to interfere with gut colonization like GA, use of broad spectrum antibiotics, severity of disease, etc. were taken into account [START_REF] Stoll | Very low birth weight preterm infants with early onset neonatal sepsis: the predominance of gram-negative infections continues in the National Institute of Child Health and Human Development Neonatal Research Network, 2002-2003[END_REF][START_REF] Gewolb | Stool microflora in extremely low birthweight infants[END_REF][START_REF] Jaureguy | Effects of intrapartum penicillin prophylaxis on intestinal bacterial colonization in infants[END_REF][START_REF] Westerbeek | The intestinal bacterial colonisation in preterm infants: a review of the literature[END_REF][START_REF] Tapiainen | Dynamics of gut colonization and source of intestinal flora in healthy newborn infants[END_REF][START_REF] Mackie | Developmental microbial ecology of the neonatal gastrointestinal tract[END_REF][START_REF] Edwards | Intrapartum antibiotic prophylaxis 1: relative effects of recommended antibiotics on gram-negative pathogens[END_REF][START_REF] Duman | Beta-lactam antibiotic resistance in aerobic commensal fecal flora of newborns[END_REF]. We believe that such an approach enabled us to characterize adequately the true differences of these two antibiotic regimens on bowel colonization.

In contrast to the previous EOS study comparing amoxicillin plus cefotaxime regimen to penicillin plus tobramycin, as well as some studies on intrapartum antibiotic prophylaxis, we failed to prove our main hypothesis that ampicillin as an antibiotic with broader Gram-negative coverage than penicillin will select for AR Enterobacteraceae except for the greater CD of AR Serratia spp [START_REF] De Man | An antibiotic policy to prevent emergence of resistant bacilli[END_REF][START_REF] Bizzarro | Changing patterns in neonatal Escherichia coli sepsis and ampicillin resistance in the era of intrapartum antibiotic prophylaxis[END_REF]. With this our findings are in accordance with Jaureguy et al. who, in an intrapartum prophylaxis study, failed to demonstrate that amoxicillin selects for betalactam resistant enterobacteria [START_REF] Jaureguy | Effects of intrapartum penicillin prophylaxis on intestinal bacterial colonization in infants[END_REF]. At present in most countries all Enterobacteraceae but E. coli have become uniformly resistant to ampicillin [START_REF] Mitt | Epidemiology of nosocomial bloodstream infections in Estonia[END_REF][START_REF] Bennet | The fecal microflora of 1-3-month-old infants during treatment with eight oral antibiotics[END_REF], and thus it is not surprising that ampicillin and penicillin have similar influence on gut colonization with Gram-negative bacteria. Still, one should bear in mind that our study was conducted in a country with low antibiotic resistance [START_REF] Loivukene | The comparison of susceptibility patterns of Gram-negative invasive and noninvasive pathogens in Estonian hospitals[END_REF] including low prevalence of extended spectrum beta-lactamase (ESBL) producing microorganisms in the PICU setting (approximately 2%) [START_REF] Mitt | Epidemiology of nosocomial bloodstream infections in Estonia[END_REF]. Future studies should identify whether these data also apply to countries with high resistance rates. A recent study from Brazil found prior penicillin use in 87% of patients colonized with ESBL producing K. pneumoniae whereas the rate among non-colonized patients was 29%; the respective data for ampicillin were 13% and 9% but the study was too small to be conclusive [START_REF] Cassettari | Risk factors for colonisation of newborn infants during an outbreak of extendedspectrum beta-lactamase-producing Klebsiella pneumoniae in an intermediate-risk neonatal unit[END_REF].

We demonstrated that differences between penicillin and ampicillin are mainly seen in colonization by Gram positive microorganisms. The association between antibiotic use other than amoxicillin and increased enterococcal colonization is well documented in literature [START_REF] Bennet | The fecal microflora of 1-3-month-old infants during treatment with eight oral antibiotics[END_REF][START_REF] Fraser | Enterococcal infections: eMedicine Specialties[END_REF][START_REF] Miedema | Risk factors for colonization with enterococci in a neonatal intensive care unit[END_REF]. The results of our study with increased colonization by enterococci in the penicillin compared with the ampicillin arm affirm this. However, which antibiotic should be preferred in this respect is more controversial. Similarly to Hufnagel et al. we demonstrate that early mucosal colonization is associated neither with increased risk of enterococcal bacteraemia nor with increase in overall mortality [START_REF] Hufnagel | Enterococcal colonization of infants in a neonatal intensive care unit: associated predictors, risk factors and seasonal patterns[END_REF]. More interestingly, recent in vitro data indicate that some strains of E. faecalis could suppress the proliferation of intestinal pathogens and thus have a potential to prevent infection and induction of inflammation suggesting that enterococcal colonization may even be beneficial.

The shift in colonization by coagulase negative staphylococci (CoNS) so that penicillin treatment was associated with greater colonization of S. epidermidis (in multivariate analysis the p=0.07) and lower colonization by S. haemolyticus and S. hominis than ampicillin is more difficult to explain and to our knowledge has not been described before. It is likely that these different species of staphylococci compete for the colonization niche with each other but we are not aware of any studies in the subject. The different colonization pattern may still have clinical relevance as mucosal colonisation is a potential source of CoNS in invasive infections [START_REF] Costa | Mucosa or skin as source of coagulase-negative staphylococcal bacteraemia?[END_REF]. Differences in the pathogenicity of CoNS species have been demonstrated with 71% of S. epidermidis but only 35% of S. haemolyticus and 26% of S. hominis strains being capable of slime production, the latter being associated with invasive disease [START_REF] Drozenova | Characteristics of coagulasenegative staphylococci isolated from hemocultures[END_REF][START_REF] Koksal | Antibiotic resistance patterns of coagulase-negative staphylococcus strains isolated from blood cultures of septicemic patients in Turkey[END_REF][START_REF] Arslan | Slime production and antibiotic susceptibility in staphylococci isolated from clinical samples[END_REF]. The relevance of differences in gut colonization observed in this study is further supported by the clinical findings showing a trend towards greater prevalence of LOS caused by S. epidermidis in the penicillin compared with the ampicillin arm (7.6 vs 2.7 per 100 patient days, respectively).

Several limitations of the study have to be noted. First, the open label design and involvement of two units only did not allow accounting for the variability potentially occur-ring in multicenter settings. Second, we did not include all admitted patients but only those who required treatment with ampicillin or penicillin. Third, the duration of each study regimen over eight months is likely too short to identify changes in the circulating microflora within a unit. Fourth, in both arms neonates received additional broad spectrum, which potentially could interfere with gut colonization; but on the other hand, this closely mirrored the clinical scenario in a PICU where the majority of infected neonates receive more than one antibiotic regimen during their stay [START_REF] Stoll | Very low birth weight preterm infants with early onset neonatal sepsis: the predominance of gram-negative infections continues in the National Institute of Child Health and Human Development Neonatal Research Network, 2002-2003[END_REF][START_REF] Westerbeek | The intestinal bacterial colonisation in preterm infants: a review of the literature[END_REF][START_REF] Schwiertz | Development of the intestinal bacterial composition in hospitalized preterm infants in comparison with breast-fed, fullterm infants[END_REF][START_REF] Mammina | Surveillance of multidrug-resistant gram-negative bacilli in a neonatal intensive care unit: prominent role of cross transmission[END_REF][START_REF] Mackie | Developmental microbial ecology of the neonatal gastrointestinal tract[END_REF]. Exclusion of these patients from the analysis would most likely introduce a major population bias with the sickest babies at highest risk of adverse colonization being left out. We believe that the use of other antibiotics did not impact the overall conclusions as in both treatment arms their frequency as well as type was similar. In addition, risk factor adjusted multivariate analysis eliminated the potential influence of confounders including broad spectrum antibiotics as much as possible. Finally, we did not evaluate quantities of colonizing microorganisms. However, the stool samples from extremely premature babies for RT-PCR analysis were collected and will be reported separately.

In conclusion, the interference with the initial gut colonization should not be a limiting factor in choosing between ampicillin and penicillin for the empiric treatment of EOS, although one should bear in mind that some clinically less important differences between these two agents occur. The selection should be based mainly on the local distribution of EOS causing microorganisms and their antibiotic susceptibility. Further studies should clarify our understanding and clinical relevance of the differences in early gut colonization by various CoNS species and enterococci.

  Fig. 1 Study outline and reasons for exclusion. Reasons for exclusion are shown on diagram as follows: A age on admission more than 72 h, B no need for early empirical antibiotic treatment, C different antibiotic regimen for more than 24 h, D need for different antibiotic regimen on admission, E transfer from neonatal intensive care unit within 24 h, F no samples. Number of cases shown on the y axis

Fig. 2 a

 2 Fig. 2 a. Colonization with Gram-positive microorganisms over time. Continuous lines present data of the penicillin and dotted lines of the ampicillin group. CoNS are represented by squares, yeasts by triangles, S. aureus by circles, and Enterococcus spp by diamonds.The following significant differences were observed: the rate of patients colonised with Enterococcus spp was greater in the penicillin as compared with ampicillin arm on days 6-9 (OR=2.99; CI 95% 1.23-7.25) and days 13-16 (OR=3.33; CI 95% 1.29-8.63). The rate of patients colonised with S. aureus on days 3 to 5 was greater in the penicillin than in the ampicillin arm (p=0.012). b. Colonization with Gram-negative and Gram-negative AR microorganisms. Continuous lines present data of the penicillin and dotted lines of the ampicillin group. Enterobacteriaceae are represented by squares, AR Enterobacteriaceae by triangles, Acinetobacter spp by circles, and AR Acinetobacter spp by diamonds. The following significant differences were observed: the rate of patients colonized by Acinetobacter spp in the penicillin as compared with the ampicillin arm on days 13-16 (6 vs 0; p=0.009) and AR Acinetobacter spp on days 10-12 (6 vs 0; p=0.045) and days 13-16 (4 vs 0; p=0.046)

Table 1

 1 Demographic data and clinical characteristics of the study population On the other hand in unit B more patients received breast milk containing regimen within the first week of life (57 vs 14; OR=6.36; 95% CI 3.33-12.17) than in unit A.

	Description	Amp + gentamicin (n=139)	Pen + gentamicin (n=137)	p
	Neonatal factors
	Duration of PICU stay days, median (IQR)	6 (3-14.8)	7.5 (4-17.2)	0.263
	GA (week) mean (±SD)	31.2 (±5.1)	31.4 (±5.1)	0.554
	GA<28 weeks, n (%)	47 (33.8)	45 (33.1)	0.996
	GA≥37 weeks, n (%)	27 (19.4)	28 (20.1)	0.952
	BW (g) mean (±SD)	1952 (±1123)	1824 (±1038)	0.946
	≤1000 g, n (%)	36 (25.9)	39 (28.4)	0.731
	≤1500 g, n (%)	72 (51.8)	70 (51.1)	0.997
	≥2500 g, n (%)	36 (25.9)	31 (22.3)	0.622
	Male/female, n	76/63	81/56	0.532
	Nutritional habits, n (%)
	Total parenteral nutrition	28 (20.1)	30 (21.9)	0.834
	Breast milk containing regimen	32 (23)	39 (28.5)	0.37
	Formula	79 (56.8)	67 (48.2)	0.231
	a Additional AB, n (%)	41 (30.9)	55 (40.1)	0.083
	Sepsis, n (%)
	Early onset sepsis	6 (4.3)	8 (6.5)	0.763
	Late onset sepsis	25 (17.9)	29 (21.2)	0.607
	Maternal factors
	Multiple births, n (%)	34 (24.5)	23 (16.5)	0.154
	Caesarean section, n (%)	79 (56.8)	78 (56.8)	0.917
	Antenatal AB, n (%)	36 (25.9)	25 (18.2)	0.166
	Antenatal steroids, n (%)	85 (61.2)	70 (50.4)	0.118
	Maternal chorionamnionitis, n (%)	21 (15.1)	29 (20.9)	0.250
	Prolonged rupture of membranes >18 h, n (%)	24 (17.3)	27 (19.4)	0.713

PICU paediatric intensive care unit, BW birth weight, GA gestational age, AB antibiotic, Amp ampicillin, Pen penicillin, IQR interquartile range a Only patients with AB treatment for more than 12 h

Table 2

 2 -4.62) and Serratia spp (OR=4.81; 95% CI 1.75-13.21), but significantly less patients harbored E. cloacae (OR=0.51; 95% CI 0.29-0.90), K. pneumoniae (OR=0.20; 95% CI 0.09-0.43), S. haemolyticus (OR=0.13; 95% CI 0.06-0.26), and S. hominis (OR=0.28; 95% CI 0.11-0.71), compared to unit A. Colonization with the Use of additional broad-spectrum antibiotics and antifungals

	Table 3 Bacterial aetiology of early and late onset sepsis by	Microorganisms	EOS episodes, n		LOS episodes, n
	treatment group
			Amp	Pen	Amp	Pen
		Gram-positive	4	4	17	28
		Staphylococcus epidermidis	-	3	5	1 4
		Staphylococcus haemolyticus	-	-	7	4
		Staphylococcus hominis	-	1	-	1
		Other CoNS	-	-	-	1
		Staphylococcus aureus: MSSA	-	-	1	2
		MRSA	-	-	1	3
		Enterococcus spp.	-	-	3	1
		Streptococcus agalactiae	4	-	-	-
		Streptococcus salivarius	-	-	-	1
		Gram-negative	2	3	13	13
		Acinetobacter baumannii	-	-	4	3
		Escherichia coli	1	1	2	1
		Enterobacter cloacae	1	1	2	2
	EOS early onset sepsis, LOS late onset sepsis, Amp ampicillin	Klebsiella oxytoca Klebsiella pneumoniae	--	--	-5	3 1
	plus gentamicin regime, Pen	Pseudomonas spp.	-	-	-	1
	penicillin plus gentamicin	Stenotrophomonas spp.	-	-	-	1
	regime, CoNS coagulase negative staphylococci, MSSA methicillin susceptible	Serratia spp. Haemophilus influenzae	--	-1	--	1 -
	Staphylococcus aureus, MRSA	Candida spp	-	1	3	1
	methicillin resistant Staphylococcus aureus	TOTAL episodes	6	8	33	42

Table 4

 4 Number of patients colonized and colonization duration (CD) of different microorganisms-an univariate hierarchical model corrected for participating unit and treatment period

	Microorganism	Patients		OR	95% CI	Mean colonization duration (CD) (n ± SD)	p
		Amp (n)	Pen (n)			Amp (n ± SD)	Pen (n ± SD)
	Gram-negatives	78	76	1.03	0.64-1.65	45.8 (40.7)	41.7 (38.9)	0.222
	Enterobacter cloacae	31	38	0.58	0.31-1.08	13.5 (20.9)	14.6 (21.2)	0.394
	Klebsiella pneumoniae	30	17	1.9	0.85-4.25	14.3 (22.7)	6.5 (11.4)	0.004
	Klebsiella oxytoca	19	22	1	0.45-2.24	6.1 (10.5)	9.8 (16.5)	0.088
	Escherichia coli	17	24	0.78	0.39-1.56	10.3 (18.2)	14.7 (24.3)	0.138
	Serratia spp	11	14	0.54	0.19-1.58	5.8 (10.6)	4.5 (8.1)	0.268
	Acinetobacter spp	9	14	0.51	0.19-1.32	2.7 (5.0)	3.8 (2.3)	0.239
	All nonfermentative	24	28	0.62	0.26-1.48	7.3 (12.1)	8.4 (13.3)	0.332
	Gram-positives	108	114	0.7	0.39-1.28	67.8 (35.6)	72.0 (29.5)	0.178
	All CoNS	97	107	0.67	0.39-1.14	56.3 (38.4)	63.9 (35.0)	0.053
	Staphylococcus epidermidis	57	69	0.69	0.43-1.1	26.3 (38.0)	34.8 (41.7)	0.039
	Staphylococcus haemolyticus	43	25	2.22	1.2-4.12	21.5 (40.0)	9.3 (23.2)	0.001
	Staphylococcus hominis	22	4	6.46	2.12-19.67	7.0 (21.2)	1.2 (8.1)	0.001
	Enterococcus spp	36	55	0.5	0.3-0.83	14.0 (20.8)	36.9 (32.6)	0.001
	Staphylococcus aureus	6	16	0.34	0.13-0.91	1.9 (3.7)	3.5 (6.4)	0.054
	Streptococcus spp	12	11	1.11	0.45-2.7	5.7 (10.4)	3.3 (6.0)	0.118
	Candida spp	26	20	1.29	0.64-2.61	12.3 (20.0)	7.7 (13.2)	0.077
	Ampicillin resistant strains
	Gram-negatives	58	55	1.06	0.66-1.7	30.2 (36.1)	27.5 (33.4)	0.285
	Enterobacter cloacae	21	19	1.09	0.49-2.42	7.3 (12.4)	5.3 (9.2)	0.169
	Klebsiella pneumoniae	21	12	1.46	0.62-3.44	6.9 (11.8)	5.2 (9.5)	0.211
	Klebsiella oxytoca	17	16	1.24	0.5-3.1	4.6 (8.0)	6.8 (12.0)	0.158
	Escherichia coli	4	7	0.66	0.18-2.38	2.5 (4.8)	3.9 (7.4)	0.249
	Serratia spp	7	4	2.43	0.41-14.18	3.9 (7.5)	0.5 (1.0)	0.011
	Acinetobacter spp	0	8	0	0.008 a	0	2.3 (4.3)	0.004
	All nonfermentative	6	13	0.39	0.07-2.05	0.8 (1.5)	3.5 (6.4)	0.354

CoNS coagulase negative staphylococci, CD colonization duration (colonizing days per 100 PICU days)

Eur J Clin Microbiol Infect Dis (2010) 29:807-816

Acknowledgements

The study was supported by Estonian Science Foundation Grant number 6984; Estonian Target Financing number 2726 and ESPID Small Grant Award. The authors are grateful to colleagues from both participating NICUs for their support and help in documenting all clinical decisions, as well as to head nurse Tiina Kilg and all nurses for their help in collecting samples. We would like to thank associated Professor Reet Mändar for her valuable remarks and suggestions while preparing the manuscript.


S Herrera-León

R González-Sanz

I Rodríguez

M R Rodicio

M A Echeita

Spread of a multiresistant CTX-M-9-producing Salmonella enterica serotype Virchow phage type 19 in Spain

Spread of a multiresistant CTX-M-9-producing serotype

Virchow phage type 19 in Spain

Introduction

Antimicrobials, specifically extended-spectrum-cephalosporins and fluoroquinolones, are often essential for the treatment of invasive infections caused by Salmonella spp. However, it is well known that these organisms have been successful at acquiring resistance to a broad range of commonly used antimicrobials, including those previously described [START_REF] Bertrand | Clonal emergence of extendedspectrum β-lactamase (CTX-M-2)-producing Salmonella enterica serovar Virchow isolates with reduced susceptibilities to ciprofloxacin among poultry and humans in Belgium and France (2000 to 2003)[END_REF]. The prevalence of multidrug-resistant (MDR) phenotypes has increased in developed countries mostly due to the mobility of antimicrobial-drug-resistant determinants, mediated by genetic elements such as integrons, transposons and plasmids [START_REF] Mazel | Integrons: agents of bacterial evolution[END_REF]. An important factor associated with this increase among particular Salmonella serotypes is the national and international spread of certain clonal genotypes, such as the global epidemic spread of MDR S. Typhimurium DT104 or the spread in the USA of the MDR strain of ceftriaxone-resistant S. Newport. Salmonella spp. accounts for more than 2,500 serotypes. About 2000 are capable of infecting humans, but only a few have assumed great significance. S. Virchow ranks third in many European countries, although it accounts for <4% of all non-typhoidal Salmonella serotypes [START_REF] Weinberger | Salmonella enterica serotype Virchow: epidemiology, resistance patterns and molecular characterisation of an invasive Salmonella serotype in Israel[END_REF]. A S. Virchow phage type 19 (PT19) strain producing a CTX-M-9 βlactamase was detected in our laboratory in 2002. In order to assess the current presence and spread of this organism and to determine its resistance properties, the present work screened a large collection of S. Virchow strains isolated from 2002 to 2006 in Spain.

Materials and methods

Strains and phage typing

The Spanish National Reference Laboratory for Salmonella (SNRLS) received 504 S. Virchow out of 39,045 Salmonella strains isolated from 2002 to 2006. Phage typing was performed according to Chambers et al. [START_REF] Chambers | A phage-typing scheme for Salmonella Virchow[END_REF], with phages and interpreting criteria provided by the Health Protection Agency (HPA, formerly Public Health Laboratory Service, Colindale) in London, England. Only S. Virchow PT19 isolates with no epidemiological link among them were selected for further studies.

Antimicrobial testing

The S. Virchow PT19 isolates were checked for susceptibility by the disk diffusion method on Mueller-Hinton agar to the following antimicrobial drugs: ampicillin (Amp, 10 µg), cephotaxime (Ctx, 30 μg), amoxicillin/ clavulanic acid (AMC, 30 µg), streptomycin (S, 10 µg), kanamycin (K, 30 µg), gentamicin (Gen, 10 µg), chloramphenicol (Cm, 30 µg), tetracycline (T, 30 µg), nalidixic acid (Na, 30 µg), ciprofloxacin (Cip, 5 µg), sulphonamides (Su, 300 µg) and trimethoprim/sulphamethoxazole (SxT, 25 μg). The breakpoints used were those defined by the Clinical Laboratory Standards Institute (CLSI) for Enterobacteriaceae [START_REF]Performance standards for antimicrobial susceptibility testing[END_REF]. Extended-spectrum βlactamase (ESBL) phenotype was detected by the double disk synergy test (DDST).

β-lactamases detection

Crude extracts from the 79 isolates with a positive DDST were obtained by sonication followed by centrifugation, and isolectric focusing was performed with a PhastSystem apparatus (GE HealthCare, Barcelona, Spain) using Phast-Gel IEF gels with gradient 3-9. Confirmation of the presence and type of an ESBL gene was done by polymerase chain reaction (PCR) and sequencing [START_REF] Rodríguez | Class 1 integrons in multidrug-resistant non-typhoidal Salmonella enterica isolated in Spain between 2002 and 2004[END_REF].

Pulsed-field gel electrophoresis

Clonality among ESBL-producing isolates was assessed by pulsed-field gel electrophoresis (PFGE) following the standard protocol of PulseNet Europe (http://www.pulseneteurope.org/docs.htm). Fingerprints were compared with InfoQuest v.4.5 (BioRad Laboratories, Hercules, CA, USA). Pattern clustering was performed by using the unweighted pair group method with an arithmetic mean and the Dice coefficient with a tolerance of 0.5%. Fragments smaller than 30 kb were disregarded according to PulseNet guidelines for standardisation. Isolates with electrophoretic patterns that differed by one band were assigned to different PFGE types.

Detection of resistance determinants

Nineteen strains (V1 to V19) were selected for further characterisation based on differences on antimicrobial resistance profiles and/or different pulse types. Detection and characterisation of class 1 integrons, gene cassettes within the variable regions, specific sequences for In60, additional resistance genes compatible with the different phenotypes and study of the quinolone resistance-determining region of gyrA of nalidixic acidresistant isolates were performed as described previously [START_REF] Rodríguez | Class 1 integrons in multidrug-resistant non-typhoidal Salmonella enterica isolated in Spain between 2002 and 2004[END_REF][START_REF] Sabaté | Novel complex sul1-type integron in Escherichia coli carrying bla CTX-M-9[END_REF][START_REF] Baucheron | The AcrB multidrug transporter plays a major role in high-level fluoroquinolone resistance in Salmonella enterica serovar typhimurium phage type DT204[END_REF].

Conjugation assays and plasmid analysis

Conjugation assays were performed with a rifampicinresistant recipient, Escherichia coli K12 J53, using in-broth methods [START_REF] Rodríguez | Class 1 integrons in multidrug-resistant non-typhoidal Salmonella enterica isolated in Spain between 2002 and 2004[END_REF]. PFGE with S1 nuclease digestion of whole genomic DNA was used to detect large plasmids in S. Virchow strains and their transconjugants [START_REF] Rodríguez | Class 1 integrons in multidrug-resistant non-typhoidal Salmonella enterica isolated in Spain between 2002 and 2004[END_REF]. Both XbaI and S1 fragments from S. Virchow and their transconjugants were transferred onto nylon membranes and hybridised with PCR-generated probes of 863 bp and 1,597 bp, specific for the bla CTX-M-9 gene and the variable region of a complex class 1 integron detected in the present study, respectively [START_REF] Rodríguez | Class 1 integrons in multidrug-resistant non-typhoidal Salmonella enterica isolated in Spain between 2002 and 2004[END_REF]. PCR-based replicon typing was performed as described by Carattoli et al. [START_REF] Carattoli | Identification of plasmids by PCR-based replicon typing[END_REF] using DNA from the transconjugants as the template.

Results and discussion

Of a total of 504 S. Virchow strains received by the SNRLS during 2002 to 2006, 99 strains isolated from sporadic cases (19.64%) were PT19. They were submitted from 14 out of the 17 Spanish provinces. All 99 strains were susceptible to amoxicillin/clavulanic acid and ciprofloxacin. A total of 90.2% isolates were resistant to ampicillin, 85.9% to cefotaxime, 62% to kanamycin, 84.8% to streptomycin, 2.2% to gentamycin, 91.3% to nalidixic acid, 73.9% to tetracycline, 3.3% to cloramphenicol, 87% to sulphonamides and 87% to cotrimoxazole. Seventy-nine out of 99 S. Virchow PT19 isolates showed antimicrobial susceptibility patterns compatible with ESBL production. Multiple drug resistance (resistance to ≥3 groups of drugs) was observed in all ESBL-producing isolates except one. The different antimicrobial resistance phenotypes were: AmpCtxKSTSuSxTNa (42 strains), AmpCtxSTSuSxTNa (16 strains), AmpCtxKSSuSxTNa (9 strains), AmpCtxS-SuSxTNa (6 strains), AmpCtxKTSuSxTNa (4 strains), AmpCtxSTSuSxT (1 strain) and AmpCtxNa (1 strain). Eleven different pulse types (P1 to P11) were observed among the 79 ESBL-producing strains. The genetic similarity was 89%. All isolates except one (V19, Table 1) were highly related (similarity ≥91%), which may indicate clonal spread of those resistances. Forty-nine strains (62%) belonged to P1. The most common antimicrobial profile within this pulse type, widely distributed over Spain, was AmpCtxKSTSuSxTNa (Table 1). Isoelectric focusing showed that all isolates produced two β-lactamases with isoelectric points of 5.4 and 7.9 except one (V1, Table 1), which only produced the second one. PCR and sequencing techniques confirmed that these values corresponded to TEM-1 and CTX-M-9 β-lactamases, respectively. Nineteen strains (V1 to V19) were selected for further characterisation based on differences on antimicrobial resistance profiles and/or different pulse types. The resistance genotypes of those strains are shown in Table 1. Apart from V16, all isolates contained a class 1 integron with the variable region (dfrA16-aadA2) and three other regions (orf513, orf3-like and orf1005) characteristic of In60, where the bla CTX-M-9 is located [START_REF] Sabaté | Novel complex sul1-type integron in Escherichia coli carrying bla CTX-M-9[END_REF]. The sequences of the variable regions and of orf513 were 100% and 99% identical to the corresponding sequences of In36 and In60, respectively, while the sequences of orf3-like and orf1005 (not present in the In36 integron) were 100% identical to the equivalent regions of In60 (GenBank accession number AF174129). In all parental strains except three (V1, V7 and V10; Table 1), plasmid DNA molecules, ranging in size from 50 to 360 kb, were visualised by S1 PFGE. E. coli transconjugants were obtained for all of the isolates tested except four (V5, V9, V12 and V19; Table 1). The transconjugants exhibited the same antimicrobial profile as their parental S. Virchow strains, except for resistance to nalidixic acid. Transconjugants harboured the same plasmids as their donor strains, with the exceptions of V1, V7, V10 and V18. Furthermore, in V1, V7 and V10, three plasmids (330, 275 and 330 kb, respectively) could only be detected after conjugation. The donor strain V18 presented two plasmids. One of them, of size 240 kb, hybridised with the bla CTX-M-9 and class 1 integron probes, whereas the other, of size 50 kb, did not hybridise with any probe. Nevertheless, the transconjugant presented a single plasmid of about 300 kb that hybridised with both probes. This size is consistent with a fusion of the two plasmids found in the donor strain. Probes for the variable region of the class 1 integron and for bla CTX-M-9 mapped on large extrachromosomal fragments detected by S1 PFGE in the strains and/or their transconjugants, with the single exception of isolate V16, which was negative for the class 1 integron (Fig. 1, panel B; Table 1). Similarly, both probes mapped together on XbaI fragments of all parental strains except for V16 that failed to hybridise with the integron probe (Fig. 1). Differences in sizes between XbaI and S1 hybridising fragments could be explained by the presence of two or more XbaI restriction sites within the plasmids.

S. enterica isolates harbouring ESBLs have emerged worldwide as being the CTX-M type which is of particular importance. Although the number of isolations of S. Virchow is low compared to those of S. Enteritidis or S. Typhimurium, the increase in resistance to several key antimicrobials is concerning. Since S. Virchow is a serotype with a predilection for extra-intestinal spread, such resistance could have therapeutic implications. S. Virchow CTX-M-9producer has been already reported from both human and poultry origin in Spain and France, suggesting a relationship among animal and human bla CTX-M-9 -containing isolates [START_REF] Riaño | Occurrence of extended-spectrum β-lactamase-producing Salmonella enterica in northern Spain with evidence of CTX-M-9 clonal spread among animals and humans[END_REF][START_REF] Weill | Emergence of extended-spectrumβ-lactamase (CTX-M-9)-producing multiresistant strains of Salmonella enterica serotype Virchow in poultry and humans in France[END_REF]. The genetic environment of this β-lactamase consists of a complex integron called In60 which is playing an active role in the dissemination of bla CTX-M-9 in different Enterobacteriaceae, including S. enterica. This genetic element was first described in E. coli, which has largely been studied since García et al. [START_REF] García | Acquisition and diffusion of bla CTX-M-9 gene by R478-IncHI2 derivative plasmids[END_REF]. The typical structure of In60 was detected in all isolates except V16, the only strain negative for the amplification of a class 1 integron. This result is supported by our hybridisation results, where only the bla CTX-M-9 probe gave a positive result, suggesting a deletion of In60. The donor strains V1, V7 and V10 did not present any plasmids by S1 digestion, although hybridisation of the XbaI fragments with the bla CTX-M-9 and class 1 integron probes were positive. Plasmids of 330, 275 and 330 kb, respectively, were observed after conjugation, and hybridisation of the S1 profiles using both probes was also positive. These results suggest a chromosomal location of the In60 integron and the mobilisation of this integron after conjugation. All transconjugants were positive for HI2 replicon except that of V18, which was also positive for FIB, supporting the hypothesis of plasmid fusion. The characterisation of S. Virchow by phage typing, PFGE, resistance properties and analysis of plasmids highlight the emergence of a multiresistant CTX-M-9-producing S. Virchow PT19 clone in Spain. This seems to be linked to the spread of closely related plasmids that share the HI2 replication system and are in the same niche of the β-lactamase, In60, and differ in additional resistance genes. Although S. Virchow PT19 CTX-M-9-producer has been reported in previous studies [START_REF] Riaño | Occurrence of extended-spectrum β-lactamase-producing Salmonella enterica in northern Spain with evidence of CTX-M-9 clonal spread among animals and humans[END_REF][START_REF] Weill | Emergence of extended-spectrumβ-lactamase (CTX-M-9)-producing multiresistant strains of Salmonella enterica serotype Virchow in poultry and humans in France[END_REF], this is the first time that the real cover of this multiresistant strain in Spain has been investigated. Moreover, our data suggest that these plasmids are related to those reported in E. coli, where similar plasticity, including plasmid fusion and the presence of In60 on the chromosome, have been described [START_REF] García | Acquisition and diffusion of bla CTX-M-9 gene by R478-IncHI2 derivative plasmids[END_REF]. It is important to underline that these plasmids harbour multiple resistances to antibiotics widely used in animals and humans. Thus, the use of a single specific antimicrobial agent could maintain the collection of plasmids in nature, allowing the exchange of resistance genes within different bacterial species and between genera.

Fig. 1

 1 Fig. 1 Examples of the mapping of CTX-M-9 and class 1 integron on XbaI and S1 PFGE profiles of S. Virchow PT19 and their transconjugants. Fragments that hybridised with probes are marked with white arrows. Panel A. Isolates negatives for conjugations. Lanes 1 to 4: V9, V5, V19 and V12 XbaI PFGE profiles. Lanes 5 to 8: V9, V5, V19 and V12 S1 PFGE profiles. Panel B. Lanes 1 to 4: V6, V11, V16

Table 1

 1 Molecular properties of S. Virchow PT19 strains selected for further characterisation of the resistances

	Strain	Pulse type	Class 1 integron	R-phenotype/R-genotype	Plasmid size (kb)	Tc plasmid size (kb)
	V1	1	1600 dfr16, aadA2	Amp Ctx K S T Su SxT Na	NO	330
				bla CTX-M-9 aphA1 aadA2 strAB
				tetA sul1 dfr16 Ser83Phe
	V2	1	1600 dfr16, aadA2	Amp Ctx S T Su SxT Na	325	325
				bla TEM-1, CTX-M-9 aadA2 tetA
				sul1 dfr16 Ser83Phe
	V3	1	1600 dfr16, aadA2	Amp Ctx K S T Su SxT Na	260	260
				bla TEM-1, CTX-M-9 aphA1 aadA2 tetA
				sul1 dfr16 Ser83Phe
	V4	1	1600 dfr16, aadA2	Amp Ctx K S Su SxT Na	340	340
				bla TEM-1, CTX-M-9 aphA1 aadA2 strAB
				sul1 dfr16 Ser83Phe
	V5	1	1600 dfr16, aadA2	Amp Ctx K S T Su SxT Na	340	NC
				bla TEM-1, CTX-M-9 aphA1 aadA2 strAB
				tetA sul1 dfr16 Ser83Phe
	V6	1	1600 dfr16, aadA2	Amp Ctx K S Su SxT Na	330	330
				bla TEM-1, CTX-M-9 aphA1 aadA2 strAB
				sul1 dfr16 Ser83Tyr
	V7	2	1600 dfr16, aadA2	Amp Ctx K S T Su SxT Na	NO	275
				bla TEM-1, CTX-M-9 aphA1 aadA2 strAB
				tetA sul1 dfr16 Ser83Phe
	V8	3	1600 dfr16, aadA2	Amp Ctx K S T Su SxT Na bla TEM-1, CTX-M-9	320	320
				aphA1 aadA2 strAB tetA
				sul1 dfr16 Ser83Phe
	V9	3	1600 dfr16, aadA2	Amp Ctx S Su SxT Na bla TEM-1, CTX-M-9	290	NC
				aadA2 tetA sul1 dfr16 Ser83Phe
	V10	4	1600 dfr16, aadA2	Amp Ctx K S T Su SxT Na bla TEM-1, CTX-M-9	NO	330
				aphA1 aadA2 strAB tetA sul1 dfr16 Ser83Phe
	V11	4	1600 dfr16, aadA2	Amp Ctx K S Su SxT Na bla TEM-1, CTX-M-9	290	290
				aphA1 aadA2 strAB sul1 dfr16 Ser83Phe
	V12	5	1600 dfr16, aadA2	Amp Ctx K S T Su SxT Na bla TEM-1, CTX-M-9	290	NC
				aphA1 aadA2 strAB tetA sul1 dfr16 Ser83Phe
	V13	6	1600 dfr16, aadA2	Amp Ctx K S T Su SxT Na bla TEM-1, CTX-M-9	360	360
				aphA1 aadA2 strAB tetA sul1 dfr16 Ser83Phe
	V14	7	1600 dfr16, aadA2	Amp Ctx K S Su SxT Na bla TEM-1, CTX-M-9	290	290
				aphA1 aadA2 strAB sul1 dfr16 Ser83Phe
	V15	7	1600 dfr16, aadA2	Amp Ctx S T Su SxT Na bla TEM-1, CTX-M-9	300	300
				aadA2 tetA sul1 dfr16 Ser83Phe
	V16	8	-*	Amp Ctx S Su bla TEM-1, CTX-M-9 strAB sul1	340	340
	V17	9	1600 dfr16, aadA2	Amp Ctx K S T Su SxT Na bla TEM-1, CTX-M-9	340	340
				aphA1 aadA2 strAB tetA
				sul1 dfr16 Ser83Phe

Table 1

 1 (continued)

	Strain	Pulse type	Class 1 integron	R-phenotype/R-genotype	Plasmid size (kb)	Tc plasmid size (kb)
	V18	10	1600 dfr16, aadA2	Amp Ctx K S T Su SxT Na bla TEM-1, CTX-M-9	241 + 50	315
				aphA1 aadA2 strAB tetA sul1 dfr16 Ser83Phe
	V19	11	1600 dfr16, aadA2	Amp Ctx K S T Su SxT Na bla TEM-1, CTX-M-9	215	NC
				aphA1 aadA2 tetA sul1 dfr16 Ser83Phe
	*: negative results; NO: not obtained; NC: not conjugative
	Amp: ampicillin; Ctx: cefotaxime; K: kanamycin; S: streptomycin; T:tetracycline; Su: sulphonamides compound; SxT: trimethoprim
	sulphamethoxazole; Na: nalidixic acid

S. Herrera-León : R. González-Sanz : M. A. Echeita Servicio de Bacteriología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo km2, 28220 Majadahonda, Madrid, Spain I. Rodríguez : M. R. Rodicio Departamento de Biología Funcional, Área de Microbiología, Universidad de Oviedo, C/ Julian Clavería s/n, 33006 Oviedo, Spain S. Herrera-León (*) Sección de Enterobacterias, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo km2, 28220 Majadahonda, Madrid, Spain e-mail: sherrera@isciii.es

Acknowledgements The work carried out at the University of Oviedo has been supported by project FIS PI080656 of the Ministerio de Ciencia e Innovación, Spain. Irene Rodríguez is the recipient of a grant from the Fundación para el Fomento en Asturias de la Investigación Científica Aplicada y la Tecnología (FICYT-BP04-086). We are grateful to Ana Aladueña for her assistance with the phage typing of the Salmonella isolates.


Juan L Eugenio-Martin

Juan A Campos-Soria

THE ROLE OF INCOME AND THE SUBSTITUTION PATTERN BETWEEN DOMESTIC AND INTERNATIONAL TOURISM

Keywords: C35 -Discrete Regression and Qualitative Choice Models &lt, C3 -Econometric Methods: Multiple/Simultaneous Equation Models &lt, C -Mathematical and Quantitative Methods, D12 -Consumer Economics: Empirical Analysis &lt, D1 -Household Behavior and Family Economics &lt, D -Microeconomics, L83 -Sports|Gambling|Recreation|Tourism &lt, L8 -Industry income elasticities, tourism demand, bivariate probit, domestic tourism, international tourism

This paper analyses the role of income in the decision of participating in the tourism demand within one year. The tourists who are participating, can travel to domestic destinations only, abroad destinations only, or to both of them. Such substitution pattern is modelled using a bivariate probit model. The analysis is carried out to the regional level using a survey conducted in 15 European countries. In addition to the traditional socioeconomic variables, the analysis adds new variables to the outbound tourism demand modelling, such as the attributes of the place of residence. The results show that tourism demand is income elastic. However, there are marked differences in the income elasticities of the probabilities of travelling domestically or abroad. Above certain income threshold, the substitution pattern between destinations takes part. The probability of travelling domestically only remains constant, whereas the probability of travelling abroad keeps growing. Additionally, the paper proves that income elasticities vary significantly and non-linearly with income.

I. Introduction

In recent decades, the relative amount of consumption on basic goods as food or clothing has been decreasing over time in favour of other consumption alternatives, such as tourism. Many regions have seen in the increase of international tourism demand as an alternative to the traditional economic activities. Provided tourists consider the region as an attractive place to visit, tourism offers a significant opportunity for economic growth and development [START_REF] Balaguer | Tourism as a long-run economic growth factors: The Spanish case[END_REF]. Tourists need accommodation, food, transportation and entertainment services. Most of these services are labour intensive and therefore their development provides growth in terms of income and employment. Moreover, tourism sector provides a significant multiplier effect on other sectors and it contributes to the current account of the balance of payments.

Along this line, increasing inbound international tourism demand has been a worldwide common target. However, more attention is being paid to the role of domestic tourism. Encouraging domestic tourism has two positive effects for a country. On the one hand, it helps retaining the benefits within the country and on the other hand, it also may help to keep a balance between the growth of the regions within a country, transferring consumption from the richer to the poorer regions. Nevertheless, not all the countries and regions are equally able to retain or attract domestic tourists from other regions of the country.

Analysis of tourism markets is a prerequisite for formulating an adequate policy for attracting tourists. For this purpose, the authorities must identify the key variables that attract tourists to choose a destination and must also attempt to adapt the attributes of the destination to meet the tourists' requirements. However, the tourism policy cannot be based solely on the preferences of current tourists, 2 but must also take account of those people who do not travel. Understanding the reasons why people do not travel is relevant for current tourist destinations but even more for new or potential tourist destinations because they can try to attract a latent demand.

During a period of time, say a year, any individual, family or household usually faces the decision of whether or not to take holidays. This decision is what we refer to as the participation decision in tourists' demand for holidays [START_REF] Mergoupis | Holiday taking and income[END_REF]. The main purpose of this paper is to study the determinants of the decision of travelling in the country of residence and or, travelling abroad within one year period. Participation decisions have been analysed as part of multiple-stage models for estimating different purposes, such as expenditure [START_REF] Hagemann | The determinants of household vacation travel: some empirical evidence[END_REF]Van Soest and Kooreman, 1987;[START_REF] Melenberg | Parametric and semi-parametric modelling of vacation expenditures[END_REF], destination choice [START_REF] Eymann | Microeconometric models of tourists' destination choice[END_REF]Nicolau and Mas, 2005), length of stay [START_REF] Fleischer | Determinants of vacation travel among Israeli seniors: theory and evidence[END_REF] and number of trips [START_REF] Hellström | A bivariate count data model for household tourism demand[END_REF].

More specific research on the participation decision has been considered by [START_REF] Mergoupis | Holiday taking and income[END_REF] and [START_REF] Alegre | Micro-economic determinants of the probability of tourism consumption[END_REF]. All the models above show the relevance of the socioeconomic characteristics of the individuals in order to estimate the willingness to travel and amongst them, special attention is paid to the role of household income.

However, explicit distinction between participation at the domestic and international level has only been explored by Van Soest and Kooreman (1987) and Nicolau and Mas (2005). Increases of income after a certain threshold may increase the probability of taking an international holiday, but at the same time it may decrease the probability of travelling domestically. Such a substitution pattern between both alternatives requires of a simultaneous estimation of both 3 decisions as carried out by Van Soest and Kooreman 1 . Hence, rather than estimating independent binary decisions as in Nicolau and Mas, it is preferable to estimate a bivariate binary model that encompasses both decisions and allows testing for the substitutability between the two alternatives. For this purpose, this paper proposes the estimation of a bivariate probit model.

The willingness to travel, the willingness to travel domestically and the willingness to travel abroad may be affected not only by the income level of the household but also by the characteristics of the region of residence. In this sense, the substitutability between travelling domestically versus abroad may also be affected by regional attributes of the place of residence of the household such as the weather, the location of the region on the coast, the proximity of the region to an international frontier, the size of the community where the household lives, or the presence of national and or international airports near the household, among others. None of the previous studies on participation of tourism demand have considered regional attributes of the place of residence of the household. Leaving aside such regional characteristics may cause biased results in the parameter estimation of the income effect and of subsequent elasticity and probability analyses. In order to incorporate these additional determinants, this paper extends the participation analysis from the national level to the regional level. The regional level 2 has only been considered by [START_REF] Mergoupis | Holiday taking and income[END_REF], but employing dummies per region as the only regional information. Therefore, this paper provides a novel approach that estimates simultaneously the decision of participation between travelling domestically and or abroad, including the 1 Van Soest and Kooreman (1987) employ simultaneous estimation between domestic and abroad percentage of tourism expenditure in relation to the income level. 2 Eymann and Ronning (1997) also consider the regional level but their purpose of study is the destination choice within a country.

II. Economic model

The economic approach to the tourism demand can be carried out from a macrolevel or a micro-level point of view. A macroeconomic framework deals with the relationship between tourism demand and main macroeconomic variables such as GDP, exchange rates or inflation amongst many others (Syriopoulos and Sinclair, 1993;[START_REF] Song | An empirical study of outbound tourism demand in the UK[END_REF]. Despite its convenience in terms of accessing data, one of the main constraints of this approach is that it relies on the assumption that each national tourist is identical at the micro-level and that they travel to all the destinations at the same time [START_REF] Papatheodorou | Why people travel to different places[END_REF]. This paper proposes a micro-founded utility model which can incorporate socio-economic variables of the tourists [START_REF] Hagemann | The determinants of household vacation travel: some empirical evidence[END_REF]. Moreover it points out the attractiveness of the place of residence of the tourist as a key variable of the individual's decision in order to consider outbound tourism as a corner solution or not.

Under the microeconomic framework, rather than analysing an 'average consumer' per region of origin, a sample of individuals is employed. These models assume individuals possess a utility function, which determines their decisions. In this sense, an individual, given a budget constraint, will choose the destination that offers highest utility level. For simplicity of the exposition, two different stages in the economic modelling are considered: first, the participation decision, and then the destination choice between travelling domestically and or abroad. , , ,

i i i in q q q q = K .
For simplicity, at this stage, it is assumed that those individuals who decide to travel do it only once. Therefore, the utility is not affected by the number of journeys taken but by the total number of days spent on the destination. Individuals are constrained by the maximum amount of time they can spend on holidays, denoted by i T . Not all the available time for holidays can be spent at the destination because they also need to spend some time in transit between their place of origin and the destination, t . Therefore, the time availability constraint is i i d T t .

Individuals' decisions are also constrained by available income, denoted by i Y .

This can be spent on other goods q at prices p and on tourism. For simplicity, it is assumed p is a vector of 1s. Two prices are considered for tourism: transportation fare, denoted by f, and the daily price of tourism, d p , which includes for instance, those expenses in accommodation or food.

The optimal solution provides the number of days spent in tourism, [Figure 1 about here] For this purpose, it is more suitable to extend the analysis employing [START_REF] Lancaster | A new approach to consumer theory[END_REF] approach to study such decisions [START_REF] Rugg | The choice of journey destination: A theoretical and empirical analysis[END_REF]. This approach considers that the individuals can obtain different utility levels from enjoying different destinations depending on the attributes that define each possible destination. Individuals yield utility from the tourism experience, which depends on the enjoyment of the attributes of the destination themselves and the number of days spent. A set of attributes z can be enjoyed from each destination with a different intensity according to a consumption technology function, such that for each jth attribute, ( )

, j j d a z g d d =
. This function varies for each attribute depending on the nature of the attribute. In this sense, as suggested by [START_REF] Morley | A microeconomic theory of international tourism demand[END_REF], there are three different sorts of attributes that can be obtained from the tourism experience. Some attributes, as those that define the physical conditions, are enjoyed at a fixed proportion, depending on the number of days spent at the destination. An example is the enjoyment of the weather. However, other attributes are enjoyed with a different intensity as the number of days spent in the destination increases. For instance, this may be the case of the experience of enjoying a new environment. Finally, some attributes can be enjoyed by tourists at once, independently on the time spent, as for instance, visiting a particular place, such as a monument or a museum. In this case, the consumption technology can be represented for each attribute linearly. This consumption technology may be perceived differently across individuals, such as:

1 1 1 i ai ai di di z d d = + 2 2 2 i ai ai di di z d d = +
Since the enjoyment of a new environment, characteristic 2 z , can decrease with the number of days spent in the destination abroad, then a non-linear relationship may also be considered for 2 z , such that: 2

1 2 ai i ai i d = + and hence 2 2 1 2 2 i i ai i ai di di z d d d = + +
. For each destination, the enjoyability obtained from each attribute on a daily basis can be represented with dotted lines as shown in [Figure 2 about here]

Using the two attributes help to show how the preferences of the individuals can drive them to choose between both destinations. Individuals maximize utility from the attributes they enjoy in the tourism experience subject to the consumption technology and the budget constraint:

( )

Max i i j U z j
Subject to:

( )

, ji ji di ai z g d d = ( ) ( ) *

III. Econometric model

The participation decision is considered as a simultaneous choice between not travelling, travelling domestically only, travelling abroad only and travelling both domestically and abroad, all these within last 12 months. The decision of travelling abroad may not be independent of the decision of travelling in the home country. Indeed, since the available leisure time and income allocated for holidays are limited, both sorts of destinations may be substitutes. Hence, independent probit regressions could be misleading. Therefore, seemingly unrelated bivariate probit model is employed. The advantage is that it allows for correlation of the error term between both decisions.

For each individual, denoting by * d T the latent variable of the willingness to travel domestically and * a T the latent variable of the willingness to travel abroad, the bivariate probit model can be specified as:

+ = + + + =
1 if 0, d d d T T T > = * * 0 if 0, 1 if 0, a a a T T T > =
From this model, a set of probabilities may be calculated in the following way: 3 It should be noted that income is gathered as an interval variable and a quarter of the sample did not state it. In order to consider the whole sample and avoid sample selection bias, income is predicted employing two step [START_REF] Heckman | Sample selection bias as a specification error[END_REF] procedure. This is followed by double censored grouped data [START_REF] Stewart | On least squares estimation when the dependent variable is grouped[END_REF][START_REF] Bhat | Imputing a continuous income variable from grouped and income missing observations[END_REF] income prediction in order to convert the grouped data into a continuous series. variable that takes unitary value if the region is on the coast.

[ ] [ ] * * 11 Pr 1, 1 Pr 0, 0 Pr , d a d a d d d a a a p T T T T x x = = = = > > = < < = [ ] ( ) ( ) Pr , , , , , d d a a x x d d d a a a d a d a d d a a x x z z dz dz x x = < < = = [ ] ( ) * * 10 Pr 1, 0 Pr 0, 0 , , d a d a d d a a p T T T T x x = = = = > < = [ ] ( ) * * 01 Pr 0, 1 Pr 0, 0 , , d a d a d d a a p T T T T x x = = = = < > = [ ] ( ) * * 00 Pr 0, 0 Pr 0, 0 , , d a d a d d a a p T T T T x x = = = = < < = ,
( ) ( ) ( ) ( ) 0, 0 1, 0 0, 1 1, 1 ln ln , , ln , , ln , , ln ,

V. Results

Estimation

The bivariate probit estimation has the advantage of running a joint estimation between the participation decision of travelling domestically and or abroad. Hence, correcting for correlation between the two decisions. Indeed, can be tested, such that if it is different than 0, then independent probits is the right approach but if it is different than 0 then the bivariate probit is the right procedure.

Using Wald test, it shows that the null hypothesis of 0 = is rejected. Hence the bivariate probit is proved to be the appropriate approach for modelling. The estimated parameters of this model are shown in Table 1.

[Table 1 about here] As expected, income is highly significant and positively related with demand.

Income elasticities vary from one level of participation to another. Taking holidays at the national level is inelastic (0.377), whereas international tourism is relatively elastic (1.188), according to the mean values. Further analysis of the income elasticities and its non-linearity is explored below.

Although the main purpose of the paper is to estimate the role of income in the participation decision in tourism demand and destination choice between travelling domestically and or abroad, the role of the other variables is also worthwhile mentioning. As shown by [START_REF] Mergoupis | Holiday taking and income[END_REF], women are keener on participating in tourism. Older individuals participate more actively in international tourism demand. Education level is highly significant, indeed, those who have studied longer are more interested in travelling. The size of the household in terms of the number of children and adults is a significant constraint in the probabilities to travel. Those households that have children are more likely to travel at the national level than internationally. Overall, despite that marital status has proved to be insignificant, individuals who are divorced or separate are Occupation is a highly relevant determinant. The occupation that provides by far the highest probability of holiday taking is studying. At the international level, general managers and self-employed professionals are more likely to travel, in contrast to unemployed, fishermen and manual workers. Owners of their own business are also very limited in their probabilities for travelling.

The socioeconomic characteristics may reveal the probabilities of taking holidays of different types of people. Nevertheless, households with similar characteristics but located in very different environments may have different willingness to travel. The members of a household located in a popular tourist destination may not have the same willingness to travel and pay for such a trip, than those living in less popular destinations. In this sense, the attributes of the place of residence may have a role as a determinant for participating in tourism demand.

Unlike most of the socioeconomic characteristics, the attributes of the place of residence are positive or negative determinants depending on the destination choice considered. Individuals living in large communities are more likely to have participated in tourism activities. They may have better transportation facilities and accessibility than those living in isolated and small communities. The probability of having travelled is lower in those regions with good weather and located on the coast. The opposite results have been found for national and international tourism. Living in a coastal area and having a good weather increases the possibilities for practising national tourism, possibly in the same region, and decreases the chances of travelling abroad. The existence of a national airport in the region will help in taking holidays nationally, whereas an international airport increases the probability that the individual will travel

The role of income and the substitution pattern between travelling domestically and abroad

As explained above, the probabilities of choosing any alternative can be estimated. More specifically, it can be shown how each of these probabilities may change with variations on the exogenous variables. In this section, a set of figures show how destination choice varies with income.

[Figure 3 about here] Figure 3 shows the relationship between income and any of the four alternatives considered in the modelling (not travelling, travelling domestically only, travelling abroad only and travelling domestically and abroad). This figure calculates the median of each of the probabilities for a given income bandwidth.

The joint of these points is carried out using interpolation provided by nonparametric techniques. The figure shows a positive relationship between travelling and income. However, a different pattern is shown for domestic and international tourism. While the probabilities of travelling to an international destination increases with income, it is not the case of the domestic tourism. The probability of travelling to a domestic destination increases with income until wage is 1,000 euros per month. However, increases in income beyond 1,000 euros do not 17 increase the probability of travelling domestically. Indeed, for earnings of 4,000 euros and beyond, the probability starts decreasing. Overall, it seems that domestic tourism and international tourism are substitutes and that as long as income increases, international tourism is preferred to domestic tourism in choosing a destination.

Further analysis considers 3d contours of the relationship between income and the different probabilities of travelling home and abroad. This is shown in figures 4, 5, 6 and 7. These contour figures represent the bivariate density distribution between income and each of the probabilities of travelling. The darker the figure the higher the joint probability is. In this sense, a non-dark area represents a very unlikely combination of income and the probability of travelling and viceversa, the darkest area represents the bivariate mode of the distribution. An additional advantage of this representation is that it allows the analysis of the distribution of the probability of travelling conditioned to a given value of income.

[Figure 4 about here]

[Figure 5 about here]

[Figure 6 about here] [Figure 7 about here] Figure 4 shows a negative relationship between the probability of not participating in tourism demand and income. The mode is to have a probability of not travelling of around 60% and around 1,200 euros of income per month. Overall, it seems to be a linear relationship between income and the non-participation decision for all Using the same scale of figure 4, figure 5 shows a low joint probability of travelling domestically and abroad within a year. It shows that most of the individuals need to choose either travelling domestically or abroad. This may happen due to time constraints, income or weather conditions of the place of residence. Such low joint probability of travelling to both sorts of destinations provides additional interest to the analysis of the destination choice and its relationship with income. The modelling allows for further disentangling of the probabilities of travelling and its relationship with income. This is explored using figures 6 and 7, which show the joint density of income and the probabilities of travelling domestically only and abroad only respectively. Comparing figures 6 and 7, it seems clear that as income grows the probability of travelling abroad only also does, whereas the probability of travelling domestically only does not.

Once the household reaches certain income threshold, it seems that further increases in income does not affect the probability of travelling domestically only, leaving this unaltered. From the figures, it can be seen that when income is 1,000, the probability of travelling domestically only is between 10% and 20% following a normal distribution around these values, though with a longer tail on the right hand side. However, the probability of travelling abroad only is positive skew, with very low probability when income is 1,000 euros per month. Furtherly, the similar analysis can be carried out for other levels of income, such as for 2,000 and 3,000 euros per month. For a given income of 2,000 or 3,000, the probability of travelling domestically only has a similar mode of around 18%, which it is slightly higher than for a given income of 1,000. The probability of travelling abroad only when income is 2,000 follows a normal distribution with mode of 19 around 30% and for an income of 3,000, a mode of around 40%. It should be noted that the tails of this probability are also much longer than those of the probability of travelling domestically only. This analysis reveals that domestic tourism is much less elastic than international tourism.

In order to get a closer picture of the income elasticities, these are calculated at different points of income, more precisely at percentiles 1, 5, 10, 25, 50, 75, 90, 95 and 99. These elasticities are referred to the sensitivity of the probability of choosing each of the alternatives proposed in the model in relation to increases in income.

[Figure 8 about here]

[Figure 9 about here]

[Figure 10 about here]

[Figure 11 about here] Figure 8 shows the elasticity of the probability of not travelling under increases in income. It shows that for low levels of income, the elasticity of not travelling is low. In absolute value, it starts increasing once the individual has reached a threshold of around 1,500 euros per month. For levels of income above 2,500 euros, tourism demand becomes elastic and such elasticity keeps growing with income. Figure 9 shows the elasticity of the probability of travelling to both sorts of destinations within a year under increases in income. It shows that such relationship is positive and for most of the population the elasticity is above 1.

VI. Conclusions

Analysis of tourism participation requires information at a more disaggregated level than has tended to occur in the past research. This paper has shown that two main types of determinants are relevant. Socioeconomic characteristics are able to explain how much individuals can travel, whereas the attributes of the place of residence complement this information indicating how much individuals want to travel and where. None of the previous studies of this kind has considered regional attributes of the place of residence of the household as a determinant. This paper proves its relevance. Additionally, including them in the model, reduce the possible bias taken by the socioeconomic characteristics, in particular, the income variable. Moreover, the analysis shows and quantifies the relevance of the key socioeconomic determinants such as income, the size of the family, age, marital status and occupation; and the characteristics of the place of residence such as weather, proximity to the coast, proximity to an international or national airport, extension of natural areas, size of the community or the price of hotels.

Their significance varies depending on the kind of tourism destination, at the national or international level. Such knowledge is useful for understanding the origin markets and their potential for keep growing or not. This is key information for tourism destinations for the design of marketing campaigns. Moreover, this model may be used for simulating scenarios for policy making. In particular, it allows the prediction of income changes and may help in determining the reliability and stability of demand from each origin market. The paper proves that, overall, tourism demand is income elastic. Furthermore, it distinguishes elasticities of not travelling, travelling domestically only, travelling abroad only and travelling domestically and abroad. Their differences show that overall, travelling domestically only has low elasticity, whereas travelling abroad only is much more elastic. From the analysis it is clear that the elasticities vary with income and usually in a non linear way. Indeed, the paper shows the existence of income thresholds. Below or above them, the sensitivity of the demand to each destination varies, and substitution between destinations occur.

Final income results are based on the behaviour of EU-15 countries as a whole.

However, results may vary for different countries depending on determinants such as location and weather. Further analysis on this issue may show a more complete picture of the income elasticities and the substitution pattern between travelling domestically and or abroad.

= " " " ! " = = # " > " " = # " " " = " > " $ $
A set of dummy variables are created such that if they take value 1 they are considered to be within the range popularly known as "good weather", such that during month m, region r is known to have a good weather if

  the household and the regional attributes of the place of residence from a pool of 16,183 households from 226 European regions (at NUTS 2 level from EU-15).

  Participation decision in tourism demand can be studied under the neoclassical theory of consumer choice. Under this approach, each individual, i

  or to both of them.

  , two attributes are considered. One attribute, 1 z , indicates the comfortability and security at the destination and the other attribute, 2 z , represents the novelty of the experience. Relaxing the assumption stated in the previous stage, two alternative destinations can be chosen. One destination is located in the same country of residence and the other one is located abroad.

Figure 2 .

 2 Figure 2.

  Three different solutions can be obtained. Two of them represent corner solutions, such as point A, which it is attained when the individuals prefer to travel abroad only, and point C when they prefer to travel domestically only. The line segment that joins these two points represents attainable combinations of the attributes for the individuals. The set of points that lie on this line segment are possible interior solutions that happen when they decide to go to both destinations, as represented for instance, by point B in Figure2. A decrease in the price of travelling abroad allows the individual to stay longer in that destination, shifting the frontier of possibilities of consumption from AC to AD as shown in Figure 2. Such a change the individual reaches a corner solution (point D) of participating abroad only instead of previous combination of both destinations.Any optimal solution depends on the ratio of marginal utilities of the attributes and prices(Deaton and Muellbauer, 1980: 250-2). Hence, what matters are not only the possible changes in prices but also the understanding of the preferences of the individuals, i.e. utility functions. The purpose of next section is to model the preferences of the individuals in participation, taking into account the socioeconomic characteristics and the attributes of the place of residence of the individual.

  both errors d and a are joint normal with means zero, variances one, and correlation . If the coefficient of correlation 0 = then the model collapses to two separate probit models. d and a are constant terms, j SE denotes j th socioeconomic variable of the household or individual, a A denotes a th attribute of the place of residence of the individual and dj , da , aj and aa denote their associated parameters. The latent variable is defined as an index function such that:

  are considered. On the one hand, a survey conducted at the household level which incorporates the socio-economic characteristics and on the other hand, a set of regional attributes that define the place of residence of the household.The survey is based on a stratified weighted survey of 16,183 households carried out from European Union (EU-15) countries in 1997. Amongst various issues, the survey covers information concerning holidays taken and socioeconomic variables, such as income 3 , occupation, age, gender, education, number of children, number of adults, marital status and the size of the community of residence.Two different kinds of attributes are considered, tourist infrastructure and geographical attributes. Within tourist infrastructure, the existence of airports and the prices of hotels are considered. Amongst the geographical attributes, weather, size of natural reserves, and coast are included.

  International airports are two discrete variables that consider the number of commercial airports available to the individual within a radio of 60 kilometres at the national and international level respectively. Hotel prices are obtained from the World Travel and Tourism Council Hotel Price Index. From there, a sample of hotel prices of three and four stars hotels are averaged independently. Further averaging of these two prices gives a final daily hotel price index. Using this index as the daily cost of stay in a regional hotel, and dividing it by household income, gives a relative measure of hotel prices for each household.Weather is an index which combines temperature, total rainfall and the number of days of rainfall per month. Such index is explained in the Appendix and it may take any value between 0 and 12 depending on how comfortable is the weather in order to practice tourism during the year. For its construction, data is collected from World Meteorological Organization. Area of natural reserves is a continuous variable that measures the extension of natural sites in the region. This is composed by National Parks and Natural Reserves. More precisely these are regions defined as type II or type V category by IUCN (International Union for Conservation of Nature and Natural Resources). Data is collected from UNEP-WCMC (United Nations Environment Programme -World Conservation Monitoring Centre) protected areas database. Coast is considered as a dummy

  travel, whereas married couples are more likely to take a holiday.

  hotel prices in the region increase the chances of travelling abroad rather than nationally. Finally, if the region is characterised by large natural reserves, then it is likely that the individual will prefer not to travel much, especially internationally.

  . This is the traditional relationship considered in the literature.

Figures

  Figures 10 and 11 show the elasticities of the participation decision of travelling

  destinations only has a positive relationship with income, when income levels are lower than 2,500 euros per month. Above that level of income, the elasticity of travelling domestically only becomes negative and increases in absolute value very much with income. However, in Figure11the behaviour of the elasticity of travelling abroad only, shows a positive relationship with income. Comparing Figure11with Figure10, it can be seen that the elasticity is much higher in the case of travelling abroad. Moreover, it shows that the preferences of the population with high income for travelling abroad keeps growing with income, whereas it decreases the interest for travelling domestically only. It underlies the substitution pattern between travelling domestically or abroad.It should be noted that the precision of the estimation of the elasticities, as shown in the figures with the 95% confidence bands, differ. Figures 9 and 11 show narrow confidence intervals for the elasticities, whereas Figures 8 and 10 have wider bands. Such less precision presented in the estimated elasticities of the decision of not travelling at all or in the decision of travelling domestically only is due to the significance of other variables rather than income. The model has proved that the place of residence matters in the decision and that despite that income is a very important determinant for the decision of travelling, other variables, as shown in the modelling matter. In other words, a wider range of possible elasticity values for travelling domestically only makes sense because the sample considers European countries with different attributes and different willingness to travel at the domestic level. Such wide bands indicates that different countries may have different elasticities for the decision of travelling domestically only. Further analysis should be carried out to prove such , the analysis of the elasticity values proves the existence of varying and nonlinear income elasticities in all the cases.

  .C. and Sinclair, M.T. (1993) An econometric study of tourism demand -the AIDS model of United-States and European tourism in Mediterranean countries, Applied Economics, 25, 1541-1552. Van Soest, A. and Kooreman, P. (1987) A micro-econonometric analysis of vacation behaviour, Journal of Applied Econometrics, 2, 215-226. temperature of region r during month m rm TR : Total rainfall in region r during month m rm DR : Days of rainfall in region r during month m

Figure 11 .

 11 Figure 1. Participation decision

Table 1 . Bivariate probit estimate of the determinants of the participation decision of travelling domestically and abroad

 1

	1 W = . This rm

Page 28 of 39 Editorial Office, Dept of Economics, Warwick University, Coventry CV4 7AL, UK Submitted Manuscript

Editorial Office, Dept of Economics, Warwick University, Coventry CV4 7AL, UK Submitted Manuscript

Acknowledgement

Authors wish to thank the helpful comments and suggestions received from M.

Thea Sinclair and Casiano Manrique. The survey used was provided by UK Data Archive and help in data inputting by M.L. Aguilera is also appreciated. Any remaining errors are responsibility of the authors. Juan L. Eugenio-Martin wishes to thank research grant received from Fundación Universitaria de Las Palmas.