MD Sarah Timmermans
email: s.timmermans@erasmusmc.nl

J Gouke

Bonsel

Régine Pm Steegers-Theunissen

Johan P Mackenbach

Ewout W Steyerberg

Hein Raat

Henri A Verbrugh

Henning W Tiemeier

Albert Hofman

Erwin Birnie

Caspar Wn Looman

Vincent Wv Jaddoe

Eric Ap Steegers

Individual accumulation of heterogeneous risks explains perinatal inequalities within deprived neighbourhoods

Keywords: perinatal mortality, prevention, residence characteristics, pregnancy, risk factors

Dutch' figures on perinatal mortality and morbidity are poor compared to EU-standards.

Considerable within-country differences have been reported too, with decreased perinatal health in deprived urban areas. We investigated associations between perinatal risk factors and adverse perinatal outcomes in 7,359 pregnant women participating in population-based prospective cohort study, to establish the independent role, if any, for living within a deprived urban neighbourhood.

Main outcome measures included perinatal death, intrauterine growth restriction (IUGR), prematurity, congenital malformations, Apgar at 5 minutes < 7, and pre-eclampsia. Information regarding individual risk factors was obtained from questionnaires, physical examinations, ultrasounds, biological samples, and medical records. The dichotomous Dutch deprivation indicator was additionally used to test for unexplained deprived urban area effects. Pregnancies from a deprived neighbourhood had an increased risk for perinatal death (RR 1.8, 95% CI [1.1; 3.1]). IUGR, prematurity, Apgar at 5 minutes < 7, and pre-eclampsia also showed higher prevalences (P < 0.05).

Residing within a deprived neighbourhood was associated with increased prevalence of all measured risk factors. Regression analysis showed that the observed neighbourhood related differences in perinatal outcomes could be attributed to the increased risk factor prevalence only, without a separated role for living within a deprived neighbourhood. Women from a deprived neighbourhood had significantly more 'possibly avoidable' risk factors. To conclude, women from a socioeconomically deprived neighbourhood are at an increased risk for adverse pregnancy outcomes.

Differences regarding possibly avoidable risk factors imply that preventive strategies may prove effective.

Abbreviations: IUGR (intrauterine growth restriction), body mass index (BMI), Brief Symptom Inventory (BSI), anxiety (ANX), depression (DEP), sexual transmittable diseases (STD) immunoglobulin G (IgG), immunoglobulin M (IgM), adverse outcome (Ao), confidence interval (CI), relative risk (RR), estimated fetal weight (EFW)

INTRODUCTION

The 2009' results from EURO-PERISTAT II demonstrated a relatively high and persistent poor perinatal mortality rate in the Netherlands (9.8 per 1,000 total births in 2006). [START_REF]European Perinatal Health Report[END_REF] A broader comparison of maternal and neonatal outcomes revealed that the Dutch figures are among the worst in Europe. [START_REF]European Perinatal Health Report[END_REF] Additionally, considerable geographic differences exist with respect to most other perinatal health indicators. [START_REF] Agyemang | The effect of neighbourhood income and deprivation on pregnancy outcomes in Amsterdam, The Netherlands[END_REF][START_REF] De Graaf | Perinatal outcomes in the four largest cities and in deprived neighbourhoods in The Netherlands[END_REF] Perinatal mortality and morbidity are significantly more prevalent in the four largest cities with particular increased risks for pregnancies from socioeconomically deprived neighbourhoods. In some deprived neighbourhoods perinatal mortality appears to be as high as 17 / 1,000. [START_REF] De Graaf | Perinatal outcomes in the four largest cities and in deprived neighbourhoods in The Netherlands[END_REF] These observed geographic inequalities emphasise the need for improvements. In light of limited opportunities to lower perinatal mortality by therapeutic interventions, increased attention to primary and secondary preventive strategies is warranted. This requires detailed information on modifiable sociodemographic, lifestyle, obstetric, and other health-related risk factors. From comparison of these perinatal risk factors across neighbourhoods novel opportunities may emerge to reduce Dutch perinatal health inequalities. Whilst extensive regarding perinatal outcomes and interventions, the Dutch perinatal registration databases are limited regarding individual-and geographical risk factors. [START_REF] De Graaf | Perinatal outcomes in the four largest cities and in deprived neighbourhoods in The Netherlands[END_REF] The Generation R Study is a prospective cohort study from early pregnancy onwards in Rotterdam, the second largest city in the Netherlands. [START_REF] Jaddoe | The Generation R Study Biobank: a resource for epidemiological studies in children and their parents[END_REF][START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF] Its detailed data collection enables the examination of the presence of neighbourhood' related effects.

METHODS

Design

The study was embedded within The Generation R Study, a population-based cohort study from early pregnancy onwards. [START_REF] Jaddoe | The Generation R Study Biobank: a resource for epidemiological studies in children and their parents[END_REF][START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF] The study is conducted in Rotterdam, the second largest city in the Netherlands comprising about 585,000 inhabitants. [START_REF]Core data Rotterdam[END_REF] Participants were pregnant women expected to deliver between 2002 and 2006. [START_REF] Jaddoe | The Generation R Study Biobank: a resource for epidemiological studies in children and their parents[END_REF][START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF] For the present study analyses were restricted to prenatally enrolled women with a singleton pregnancy ≥ 22 weeks (n = 8,668

Pregnancy outcomes

Several overlapping sources (obstetric caregivers, ultrasound facilities, Municipal Health Services) provided information about A) location and mode of delivery (home or hospital delivery as the Dutch obstetric system is characterised by its home delivery policy , emergency or elective caesarean section, instrumental vaginal delivery) and B) pregnancy outcomes including intrauterine growth restriction (IUGR), pre-eclampsia, intrauterine fetal death, birth weight, gestational age at birth, congenital malformations, Apgar score five minutes after birth, and early neonatal death. [START_REF] Jaddoe | The Generation R Study Biobank: a resource for epidemiological studies in children and their parents[END_REF][START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF] IUGR and low birth weight were defined as a SD-score < -1.28, and prematurity as delivery < 37.0 weeks.

Last, we conveniently defined the compound measure 'Adverse Outcome (Ao)' as the presence of one or more of the following perinatal events: perinatal death, congenital malformations, prematurity, low birth weight, and/or Apgar < 7.

Individual perinatal risk factors

Sociodemographic risk factors were assessed by a survey in early pregnancy. These included maternal age, marital status, consanguinity, and measures of socioeconomic status and ethnicity.

Socioeconomic status was defined by educational level, net household income, and employment status. [START_REF] Jaddoe | The Generation R Study Biobank: a resource for epidemiological studies in children and their parents[END_REF][START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF][START_REF]Standard classification people with a foreign background[END_REF] Maternal occupation was classified as employed (paid or self-employment) or unemployed (job-seeking, social security or disability benefit, housewife, student). The number of weekly working hours was also assessed to make a further distinction. Ethnicity was defined by country of birth of the woman herself and her parents.

Neighbourhood deprivation classification

Women were categorised as residing within or outside of a deprived neighbourhood based on the postcode of their place of living which can then be converted using the published Dutch index of deprivation 2007. We acquired participants' postcodes from the Centre for Research and Statistics

Rotterdam. [START_REF]Core data Rotterdam[END_REF] The deprivation index 2007 is national indicator of neighbourhood deprivation designed by the Netherlands' Department of Housing, Spatial Planning, and the Environment. [START_REF]the Netherlands' Ministry of Housing, Spatial Planning and the Environment/ Directorate General for Housing, Communities and Integration[END_REF] The index is created from separate scores based on five domains of deprivation: housing, employment, education, integration, and safety. The index assigned the label 'deprived' to 83 out of 4,878 Dutch postcodes, of which 23 postcode-areas are located in Rotterdam.

Statistical methods

Of the eligible women 849 had ≥ 35% missing values. After exclusion of these women the sample available for final analysis was 7,359. With respect to the remaining data missing values were imputed using multiple imputation. In the present study for each missing value five draws were performed providing five substituted data which in turn created five completed data sets. Analyses were performed separately on each completed dataset and thereafter combined into one global result. [START_REF] Sterne | Multiple imputation for missing data in epidemiological and clinical research: potential and pitfalls[END_REF] The difference of adverse pregnancy outcomes between deprived and non-deprived neighbourhoods was compared using the Independent Student's t, Mann-Whitney U, and chi-square test. For all defined outcomes, we estimated the crude Relative Risk (RR) of living within a deprived neighbourhood (RR, 95% Confidence Interval [95% CI]).

Similarly, we compared between deprived and non-deprived area's the prevalence of all separate risk factors, and the prevalence of a combined score (weighted summation of all separate risks into one number representing the overall risk load). To obtain the combined score, we first derived a per risk factor weight from literature (either 0, 1, or 2; for a detailed account see Appendix 1). The combined score ranged from 0 -20, where 0 represents absence of any risk.

As a third step we applied multivariable logistic regression analysis where the occurence of any of the indicators for adverse perinatal outcome was related to all measured individual risk factors plus the deprivation indicator was added. If the individual risk factors in this dataset would explain the prevalence of adverse pregnancy outcomes and no additional role would exist for the variable 'living within a deprived neighbourhood', then the excess risk of adverse pregnancy outcome (if present) would be fully explained by the (increased) prevalence of individual risk factors only. In view of collinearity both a forward and backward regression strategy was applied for all five imputed datasets separately (P inclusion = P exclusion = 0.10). If the same association was observed across four or five datasets, this was interpreted as being significant. In that case we report on the median size of that coefficient together with its 95% CI.

Subsequently, we graphically compared the compound risk score distribution, including a comparison of the proportion women with A) low risk (sum score < 3); B) medium risk (sum score 3 -7); or C) high risk (sum score ≥ 7).

Lastly, to assess possibilities for preventive strategies, we recoded our risk factors into 'possibly avoidable' and 'possibly non-avoidable', based on the list of Rutstein et al. [START_REF] Rutstein | Measuring the quality of medical care": revision of tables of indexes[END_REF] Avoidability was judged based on the presence of evidence that the occurrence of an adverse perinatal outcome might be prevented by primary or secondary preventive measures embedded in preconception or prenatal care. This approach classified the following risk factors as 'possibly avoidable': number of

RESULTS

Data were available on 7,359 women belonging to 71 area-postcodes. Of these women 37,8% was resident within a deprived neighbourhood (n = 23 area-postcodes) and 62,2% outside of a deprived neighbourhood (n = 48 area-postcodes). Considerable differences in pregnancy outcomes were apparent across deprived and non-deprived neighbourhoods (Table 1). Overall perinatal mortality encompassing intrauterine and neonatal death was 0.7% (55 / 7,359). Women residing within a deprived neighbourhood had almost a twofold increase in risk for perinatal death as compared to women residing outside of a deprived neighbourhood (RR 1.8, 95% CI [1.08; 3.11]). This difference was mainly explained by the substantially higher number of intrauterine fetal deaths in deprived neighbourhood pregnancies (20 / 2,779 versus 17 / 4,580). Nearly all other adverse pregnancy including IUGR, pre-eclampsia, low birth weight, prematurity, and Apgar < 7, showed higher prevalences in pregnancies from deprived neighbourhoods. In contrast, the risks for hospital delivery, elective caesarean section, and instrumental vaginal were similar.

Maternal characteristics and risk factors are presented in Table 2. Forty percent of the study population was of non-Western descent (n = 2,903). The majority of these women resided within a deprived neighbourhood (1,660 / 2,779). We observed large differences in the prevalence of sociodemographic, lifestyle, obstetrical, and health-related determinants between women living within and outside of a deprived neighbourhood. Deprived neighbourhood' women were younger, had lower measures of socioeconomic status, and suffered more often from obesity, psychopathology, and STDs (all P < 0.05). We also observed higher percentages of unplanned pregnancies among these women as well as higher percentages of adverse lifestyle factors including smoking or no folic acid use.

Logistic regression (with 'Adverse Outcome' as outcome) consistently showed strong effects for the risk factors non-Western and Afro-African ethnicity, low income, BMI < 20, smoking, nulliparity, complications in previous pregnancy, and two interaction-terms (low age*single; and

Afro-African ethnicity*single) (Table 3). We observed minor differences across the five datasets as well as in the forward/backward approaches (Appendix 2). In none of these analyses, living within a deprived neighbourhood emerged as significant independent risk factor. Additional prediction of 'living within a deprived neighbourhood' from the same risk set provided the same solution (equal risk factors) as the analysis predicting 'Adverse Outcome'. Apparently, in the present study the effect of neighbourhood deprivation on perinatal outcome was explained by a differential prevalence of individual risk factors (intermediate factors).

In Figure 1 the summated risk scores for women from a deprived neighbourhood are shown (35.8% high-risk score for women from a deprived neighbourhood versus 15.1% high-risk score for women from a non-deprived neighbourhood). The figure graphically supports the key role of individual risk excess in deprived neighbourhood areas.

Lastly, Figure 2 shows that women residing within a deprived neighbourhood have significantly more 'possibly avoidable' risk factors as compared to women from of a non-deprived neighbourhood.

DISCUSSION

To our knowledge, this is the first study to examine associations between modifiable perinatal risk factors and perinatal outcomes on the neighbourhood level to enable the narrowing of perinatal inequalities in disadvantaged urban areas. Women from a deprived neighbourhood are at a substantially increased risk for adverse pregnancy outcomes, with a twofold increased risk for perinatal death. This excess risk can be attributed to an accumulation of sociodemographic, lifestyle, obstetric, and health-related risk factors present within deprived neighbourhoods. The higher proportion of modifiable risk factors in disadvantaged areas provides opportunities for primary and secondary prevention.

Key findings

Our findings on perinatal outcome inequalities within deprived neighbourhoods strengthen previous studies from Europe and the United States.[2, 3, 14-18] The majority of these studies focussed only on preterm birth and low birth weight, as important risk factors for infant mortality and morbidity. [START_REF] Gray | Social inequalities in preterm birth in Scotland 1980-2003: findings from an area-based measure of deprivation[END_REF][START_REF] O'campo | Neighborhood deprivation and preterm birth among non-Hispanic Black and White women in eight geographic areas in the United States[END_REF][START_REF] Sellstrom | Are there differences in birth weight between neighbourhoods in a Nordic welfare state?[END_REF][START_REF] Farley | The relationship between the neighbourhood environment and adverse birth outcomes[END_REF] We added pre-eclampsia, congenital anomalies, and suboptimal start at birth, since the latter two contribute to perinatal mortality, whereas pre-eclampsia is a major driver for both perinatal and maternal mortality. To our knowledge to date no study on neighbourhood deprivation and perinatal outcome has been conducted in such detail. An important finding that has not been reported before was the 50% increased risk of pre-eclampsia for women from a deprived neighbourhood, despite the inevitably higher prevalence of 'protective' smoking. Neighbourhood deprivation and cardiovascular disease are known to be strongly linked. [START_REF] Eames | Social deprivation and premature mortality: regional comparison across England[END_REF] Since pre-eclampsia has been shown to be related to cardiovascular disease, a higher prevalence of the latter may have contributed to these results. [START_REF] Bellamy | Pre-eclampsia and risk of cardiovascular disease and cancer in later life: systematic review and meta-analysis[END_REF] Interestingly, the risk for congenital malformations for women from a deprived neighbourhood was not increased. Previously, de Graaf et al. [START_REF] De Graaf | Perinatal outcomes in the four largest cities and in deprived neighbourhoods in The Netherlands[END_REF] reported similar results.

They suggested that the higher uptake of prenatal screening in urban areas, as compared to rural areas, might be causative. [START_REF] De Graaf | Perinatal outcomes in the four largest cities and in deprived neighbourhoods in The Netherlands[END_REF] Future studies are needed to investigate this finding in closer detail.

In our study accumulation of risk factors largely explained perinatal inequalities according to place of living. Although the exact underlying mechanisms through which neighbourhood deprivation influences both perinatal risk factors and outcomes are unknown, several pathways have been proposed which share that geographical and individual characteristics are closely linked. [START_REF] Culhane | Neighborhood context and reproductive health[END_REF] One model proposes that neighbourhood-characteristics affect reproductive outcome by modelling of a wide range of individual-level economic opportunities and risk behaviours. [START_REF] Culhane | Neighborhood context and reproductive health[END_REF][START_REF] Av Dr | The examination of neighbourhood effects on health: conceptual and methodological issues related to the presence of multiple levels of organisation[END_REF][START_REF] Kleinschmidt | Smoking behaviour can be predicted by neighbourhood deprivation measures[END_REF] In this model access to education and training programs is determined by place of living. Better education yields higher socioeconomic status which in turn benefits perinatal outcomes. [START_REF] Av Dr | The examination of neighbourhood effects on health: conceptual and methodological issues related to the presence of multiple levels of organisation[END_REF] On the other hand, locally shared social characteristics may influence unhealthy risk behaviours through common cultural norms and beliefs, which in turn are associated with perinatal outcomes (i.e. smoking habits, sexual behaviour). [START_REF] Culhane | Neighborhood context and reproductive health[END_REF][START_REF] Kleinschmidt | Smoking behaviour can be predicted by neighbourhood deprivation measures[END_REF] Essentially all perinatal risk factors investigated in our study have been previously reported to be associated with both adverse neighbourhood conditions and reproductive health. [START_REF] Culhane | Neighborhood context and reproductive health[END_REF][START_REF] Av Dr | The examination of neighbourhood effects on health: conceptual and methodological issues related to the presence of multiple levels of organisation[END_REF][START_REF] Kleinschmidt | Smoking behaviour can be predicted by neighbourhood deprivation measures[END_REF] During recent years increasing attention has focused on preconception care as a means of optimising women's health and knowledge before planning and conceiving pregnancy in order to reduce the risk of adverse health effects for the woman, fetus, or neonate. [START_REF]The importance of preconception care in the continuum of women's health care[END_REF][START_REF] Sheldon | Netherlands considers introducing preconception care[END_REF] Several countries have succesfully introduced structured preconception health care programmes. [START_REF]The importance of preconception care in the continuum of women's health care[END_REF][START_REF] Sheldon | Netherlands considers introducing preconception care[END_REF][START_REF] Wildschut | Preconception care: an essential part of the care for mother and child[END_REF][START_REF] Johnson | Recommendations to improve preconception health and health care--United States. A report of the CDC/ATSDR Preconception Care Work Group and the Select Panel on Preconception Care[END_REF][START_REF] Boulet | Preconception care in international settings[END_REF][START_REF] Ebrahim | Models of preconception care implementation in selected countries[END_REF][START_REF] Czeizel | Ten years of experience in periconceptional care[END_REF] One of the most effective programmes, The Hungarian Periconceptional Service, reported reductions in congenital defects and ectopic pregnancy rates, and higher birth weights. Moreover, urogenital infections were detected and treated, and screening for diabetes and cardiovascular disease led to referral of 3% of women to special clinics. [START_REF] Czeizel | Ten years of experience in periconceptional care[END_REF] The observed difference in modifiable perinatal risk factors between deprived and nondeprived neighbourhood' women provides opportunities for improvement. In this respect we consider the active provision of information as vital to reduce perinatal inequalities since most women appear to be largely ignorant on the consequences of an adverse lifestyle or of the presence of a medical condition during pregnancy, and in particular during the periconception period. [START_REF]The importance of preconception care in the continuum of women's health care[END_REF][START_REF] Hollingsworth | Expanded care in obstetrics for the 1980s: preconception and early postconception counseling[END_REF] A precondition for success of intensified care is free access to, and due start of preconception and prenatal care. Most women from a deprived neighbourhood entered antenatal care at such a late stage that it can be concluded that opportunities to improve the health of these women and their offspring were completely missed. [START_REF] Alderliesten | Late start of antenatal care among ethnic minorities in a large cohort of pregnant women[END_REF] Immigrant women and women of very low socioeconomic class require special attention because they are often not reached in the provision of information about the consequences of an adverse lifestyle or medical condition due to language, cultural, and/or social barriers. [START_REF] Ruhl | The clinical content of preconception care: preconception care for special populations[END_REF] Without extra attention, intensification of prenatal care will have a differential effect according to the place of living. [START_REF] Culhane | Neighborhood context and reproductive health[END_REF]

Strengths and weaknesses

This was a survey embedded in a large population-based prospective cohort with an extensive data collection. The use of structural variables to characterise neighbourhood deprivation rather than using aggregates of individual-level variables adds to its strength. [START_REF]the Netherlands' Ministry of Housing, Spatial Planning and the Environment/ Directorate General for Housing, Communities and Integration[END_REF][START_REF] Culhane | Neighborhood context and reproductive health[END_REF] Nevertheless, some limitations need to be discussed. First, selective participation has been demonstrated for deprived areas. [START_REF] Goodman | Who are we missing? Area deprivation and survey participation[END_REF] Though participation rates for the Generation R Study are relatively high and the ethnic distribution differs only moderately from that of the eligible population, the Generation R Study is characterised by a rather highly educated and healthy study population compared to available city data. [START_REF] Jaddoe | The Generation R Study Biobank: a resource for epidemiological studies in children and their parents[END_REF][START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF] This selective non-response towards higher socioeconomic status may have influenced the associations. [START_REF] Tromp | Anonymous non-response analysis in the ABCD cohort study enabled by probabilistic record linkage[END_REF] Moreover, we had to exclude ten percent of the study population because of high levels of missing data with subsequent considerable multiple imputations. Since these participants were predominantly lower educated and of non-western ethnicity this may have biased our results.

Lastly, we obtained most information on perinatal risk factors by questionnaires in Dutch and English.

If required individual support in Arabic, French, Portuguese, or Turkish was available. Despite these efforts, misclassification through language proficiency should always be considered.

Future research

The inequalities in the prevalence of aetiological factors urge for detailed analysis of the provision of care, in particular the presence of substandard care relative to risk exposure. In the Netherlands, recently concerns were raised on the quality of delivered perinatal care. [START_REF] Sheldon | Obstetric care must change if Netherlands is to regain reputation for safe childbirth[END_REF][START_REF] Merkus | Perinatal mortality in The Netherlands: an audit is now more necessary than ever[END_REF][START_REF] Alderliesten | Ethnic differences in perinatal mortality. A perinatal audit on the role of substandard care[END_REF][START_REF] Van Roosmalen | Substandard care in immigrant versus indigenous maternal deaths in The Netherlands[END_REF] Substandard factors in maternity care were more likely to occur in immigrant than in indigeneous women. [START_REF] Alderliesten | Ethnic differences in perinatal mortality. A perinatal audit on the role of substandard care[END_REF][START_REF] Van Roosmalen | Substandard care in immigrant versus indigenous maternal deaths in The Netherlands[END_REF] The similar prevalence of instrumental vaginal delivery and caesarean sections across deprived neighbourhoods may suggest under-treatment in view of the much higher prevalence of conditions like IUGR and pre-eclampsia. Such research should apply multiple methods: epidemiological analysis of national data, qualitative in-depth research into professional mechanisms, and perinatal audit methods such as the one currently being implemented. [START_REF] Merkus | Perinatal mortality in The Netherlands: an audit is now more necessary than ever[END_REF][START_REF] Ravelli | Decreasing perinatal mortality in The Netherlands, 2000-2006: a record linkage study[END_REF]

Conclusions

Women from a deprived neighbourhood are at almost double risk for adverse pregnancy outcomes, which can be largely attributed to accumulation of individual risk factors present within underprivileged urban areas. Targeted preconception healthcare programmes should be implemented in order to lower the geographic inequalities. results from logistic regression analysis with forward and backward approach (P inclusion = P exclusion = 0.10) for all 5 imputed datasets separately. If the same association was observed across 4 or 5 datasets, this was interpreted as a significant association. In that case we report on the combined estimate given as the median size of that coefficient together with its 95% CI. NI not included in final step

TABLES

FIGURE CAPTIONS TOGETHER WITH EXPLANATORY FOODNOTES Figure 1 DistributionFigure 2 '

 12 FIGURE CAPTIONS TOGETHER WITH EXPLANATORY FOODNOTES

  The following classification was applied:

	Dutch, Moroccan, Turkish, Cape-Verdean, Antillean, Surinamese, other non-Western, and other
	Western. Because women of Antillean or Surinamese descent are of mixed ethnic origin we further
	categorised these women into Surinamese Hindustani or Afro-African, Antillean Afro-African, and
	Antillean/Surinamese-other.[9]
	Information regarding obstetric characteristics was obtained from the same questionnaire.
	This included parity, pregnancy planning, folic acid use, obstetric history, and gestational age at
	booking.[4, 5] With respect to lifestyle factors, height and weight were measured to calculate body
	mass index at inclusion (BMI; in kg / m 2 ). Information on smoking, alcohol, and recreational drug use
	was obtained by questionnaires in early, mid-, and late pregnancy.[4, 5] Psychopathology was
	assessed using the Brief Symptom Inventory (BSI) in mid-pregnancy.[4, 5, 10] For the present study
	we used both anxiety (ANX) and depression scales (DEP) with a cut-off score ≥ 0.67. Other health
	characteristics included self-reported history of hypertension, diabetes, heart disorders,
	hypercholesterolaemia, systemic lupus erythomatosus, multiple sclerosis, or thyreoid disease (i.e.
	comorbidity), as well as sexual transmittable diseases (STD) comprising of chlamydia infection
	(measured in urine by PCR), hepatitis B (hepatitis B surface antigen measured in blood samples),
	toxoplasmosis (immunoglobulin G [IgG] and IgM Toxoplasma gonadii antibodies measured in blood
	samples), and HIV-infection (self-reported).[4, 5]

Table 1

 1 Percentages and relative risks of pregnancy outcomes according to neighbourhood classification

		Deprived neighbourhood
	Total	Yes	No	RR (95% CI)
	N = 7,359	n = 2,779	n = 4,580

Table 2

 2 Prenatal maternal characteristics according to neighbourhood classification

		Deprived neighbourhood
	Total	Yes	No
	N = 7,359	n = 2,779	n = 4,580

* * * * * * * * * * *

Median weighted risk score sum (range)

		4 (0-14)	5 (0-14)	3 (0-14)*
	High risk profile (sum score ≥ 7)	17.8	35.8	15.1*
	values represent percentage within column, gestational age at inclusion in weeks.

* P value < 0.05

FUNDING

The work was supported by the Erasmus Medical Centre Rotterdam, the Erasmus University Rotterdam, and the Netherlands Organisation for Health Research and Development (ZonMw).

Vincent WV Jaddoe also acknowledges the personal funding by the Netherlands Organization for Health Research (ZonMw 90700303)

AKNOWLEDGEMENTS

We gratefully acknowledge the contribution of general practitioners, hospitals, midwives, and pharmacies in Rotterdam

CONFLICTS OF INTEREST STATEMENTS

None to declare

ETHICAL APPROVAL

The study was approved by the Medical Ethics Committee of the Erasmus MC Rotterdam, the Netherlands.

Table 3

Association between individual risk factors and the occurrence of ' Adverse Outcome', results from five imputed datasets and forward and backward regression respectively

Forward approach

Backward approach Risk factor Significant


Grégory ; Bièvre

Denis Jongmans

David Goutaland

Vilma Zumbo

Keywords: landslide, clay, drainage, kinematics, geophysics

come

Lithological control on the kinematic pattern and the location of slip surfaces in a large clayey landslide (Trièves, French Alps)

http://mc.manuscriptcentral.com/esp Earth Surface Processes and Landforms


Alzennyr Da Silva
email: alzennyr.dasilva@inria.fr

Yves Lechevallier
email: yves.lechevallier@inria.fr

Fabrice Rossi
email: fabrice.rossi@inria.fr

Francisco De Carvalho

Clustering Dynamic Web Usage Data

Most classification methods are based on the assumption that data conforms to a stationary distribution. The machine learning domain currently suffers from a lack of classification techniques that are able to detect the occurrence of a change in the underlying data distribution. Ignoring possible changes in the underlying concept, also known as concept drift, may degrade the performance of the classification model. Often these changes make the model inconsistent and regular updatings become necessary. Taking the temporal dimension into account during the analysis of Web usage data is a necessity, since the way a site is visited may indeed evolve due to modifications in the structure and content of the site, or even due to changes in the behavior of certain user groups. One solution to this problem, proposed in this article, is to update models using summaries obtained by means of an evolutionary approach based on an intelligent clustering approach. We carry out various clustering strategies that are applied on time sub-periods. To validate our approach we apply two external evaluation criteria which compare different partitions from the same data set. Our experiments show that the proposed approach is efficient to detect the occurrence of changes.

Introduction

Web Mining [START_REF] Kosala | Web mining research: A survey[END_REF] appeared at the end of 90s and consists in using Data Mining techniques in order to develop methods that allow relevant information to be extracted from Web data (such as documents, interaction traces, link structure, etc.). A more specialized branch of this domain, called Web Usage Mining (WUM) [START_REF] Cooley | Data preparation for mining world wide web browsing patterns[END_REF] [14], deals with techniques based on Data Mining that are applied to the analysis of users behavior in a website. The present article is placed is this last context.

WUM consists in extracting interesting information from files which register Web usage traces. This activity has become very critical for effective website management. In the e-commerce domain, for example, one of the most important motivations for the analysis of usage is the need to build up consumer loyalty and to make the site more appealing to new visitors. Accurate Web usage information could help to attract new customers, retain current customers, improve cross marketing/sales, measure the effectiveness of promotional campaigns, track leaving customers and find the most effective logical structure for their Web space. Other applications we can cite are the creation of adaptive websites, support services, personalization, network traffic flow analysis, etc.

Most traditional methods in this domain take into account the entire period during which usage traces were recorded, the results obtained naturally being those which prevail over the total period. Consequently, certain types of behaviors, which take place during short sub-periods are not detected and thus remain undiscovered by traditional methods. It is, however, important to study these behaviors and thus carry out an analysis related to significant time sub-periods. It will then be possible to study the temporal evolution of users' profiles by providing descriptions that can integrate the temporal aspect. The access patterns to Web pages are indeed of a dynamic nature, due both to the on-going changes in the content and structure of the website and to changes in the users' interest. The access patterns can be influenced by certain parameters of a temporal nature, such as the time of the day, the day of the week, recurrent factors (summer/winter vacations, national holidays, Christmas) and non-recurrent global events (epidemics, wars, economic crises, the World Cup). Furthermore, as the volume of mined data is great, it is important to define summaries to represent user profiles.

WUM has just recently started to take account of temporal dependence in usage patterns. In [START_REF] Roddick | A survey of temporal knowledge discovery paradigms and methods[END_REF], the authors survey the work to date and explore the issues involved and the outstanding problems in temporal data mining by means of a discussion about temporal rules and their semantic. In addition, they investigate the confluence of data mining and temporal semantics. Recently in [START_REF] Laxman | A survey of temporal data mining[END_REF], the authors outline methods for discovering sequential patterns, frequent episodes and partial periodic patterns in temporal data mining. They also discuss techniques for the statistical analysis of such approaches. Notwithstanding these considerations, the majority of methods in WUM are applied over the entire period that covers all the available data. Consequently, these methods reveal the most predominant behaviors in data, and the interesting short-term behaviors which may occur during short periods of time are not taken into account. For example, when the data analysed is inserted into a dynamic domain related to a potential long period of time (such as in the case of Web log files), it is to be expected that behaviors evolve over time.

These considerations have given rise to many studies in data analysis, especially concerning the adaptation of traditional static data-based methods to the dynamic data framework. In this line of research, our proposition is to use summaries obtained by an evolutionary clustering approach applied over time sub-periods to carry out a follow-up of the user profile evolution. The main objective of this article is to propose and evaluate a monitoring clustering strategy which is able to find changes in a stream of Web usage data.

This article is organized as follows. Section 2 describes the proposed clustering approach based on time sub-periods. Section 3 presents the experimental framework including the benchmark data set analysed, the algorithm and the external evaluation criteria adopted as well as a discussion on the results obtained. The last section reports the final conclusion and some suggestions for future work.

Clustering approach based on time sub-periods

In unsupervised classification modelling of dynamic data, new clusters may emerge at any time and existing clusters may evolve or disappear. The clustering problem can be seen as an evolutionary process of detecting and tracking dynamic clusters. The approach proposed in this article consists initially in splitting the entire time period analysed into more significant sub-periods. In our experiments, we use the months of the year to define the time sub-periods (cf. section 3.4). This is done with the aim of discovering the evolution of old patterns or the emergence of new ones. This fact would not have been revealed by a global analysis over the whole time period. After this first step, a clustering method is applied on the data of each time sub-period, as well as over the complete period. The results provided for each clustering are then compared.

The main base of our proposition regards the specification of a compact structure to summarize the clustering of data within a time sub-period without storing the entire data set. This structure is designated by the cluster centre (prototype) and represents a user profile. These prototypes reflect the behaviour of individuals belonging to the same cluster.

The following subsections describe in detail the four clustering strategies we study.

Global clustering

This clustering strategy corresponds to the traditional practice, that is to say, the clustering algorithm is applied on the entire data set containing all the individuals, without taking into account the temporal information. After applying the clustering algorithm, we have a partition containing K clusters. We then apply a filter on each of these clusters in order to define new sub-clusters containing individuals belonging to the same time sub-periods (for example, the same month of the year) (cf. figure 1). After that we put together subclusters belonging to the same time sub-periods, which defines one partition per sub-period. We will then compare these last partitions with those obtained by the other clustering strategies.

Independent local clustering

In this clustering strategy, we first split the data set by time sub-period. After that, we apply the clustering algorithm on each sub-period individually, which gives us as many partitions as there are time sub-periods. At the end of this process we will have a partition containing K clusters in each time sub-period, each independent from the other. (cf. figure 2).

Previous local clustering

In this clustering strategy, we begin by applying the clustering algorithm in the first time sub-period, which gives us a partition into K clusters. We take the prototype of each of these clusters in order to define a new partition on the individuals belonging to the next time sub-period. To do so, we apply the Fig. 2. Independent local clustering clustering allocation phase. We repeat this process for all the following time sub-periods (cf. figure 3).

Dependent local clustering

This clustering strategy has some points in common with the previous one. The main difference here is that we use the prototypes obtained by the clustering on the previous time sub-periods to initiate a completely new clustering process rather than applying only the allocation phase. In other words, we run the algorithm until its convergence. As a case study, we use a benchmark website from Brazil3 . This site contains a set of static pages (details of teaching staff, academic courses, etc.) and dynamic pages (see [START_REF] Silva | Mining web usage data for discovering navigation clusters[END_REF][3][12] [START_REF] Rossi | Dissimilarities for web usage mining[END_REF] for an analysis of this part of the site). We studied the accesses to the site from 1 st July 2002 to 31 st May 2003.

For Web usage data pre-processing, we adopt the methodology proposed by [START_REF] Tanasa | Advanced data preprocessing for intersites web usage mining[END_REF] who defines a navigation as a succession of requests not more than 30 minutes apart, coming from the same user. In order to analyse the more representative traces of usage, we selected long navigations (containing at least 10 requests and with a total duration of at least 60 seconds) which are assumed to have originated from human users (the ratio between the duration and number of requests must be at least 4, which means a maximum of 15 requests per minute). This was done in order to extract human navigations and exclude those which may well have come from Web robots. The elimination of short navigations is justified by the search for usage patterns in the site rather than simple accesses which do not generate a trajectory in the site. After filtering and eliminating outliers, we obtained a total of 138,536 navigations.

Clustering algorithm

Our method uses an adapted version of the dynamic clustering algorithm [START_REF] Anderberg | Cluster analysis for applications[END_REF][5] [START_REF] Macqueen | Some methods for classification and analysis of multivariate observations[END_REF] applied on a data table containing the navigations in its rows and real-value variables in its columns (cf. table 1). As a distance measure, we adopt the Euclidean distance. For all the experiments, we defined an a priori number of clusters equal to 10 with a maximum number of iterations equal to 100. The number of random initialisations is equal to 100, except when the algorithm is initialised with the results obtained from a previous execution (for the strategies presented in section 2.3 and section 2.4).

Evaluation criteria

To analyse the results, we apply two external criteria. This implies that we evaluate the results of a clustering algorithm based on how well the clustering matches pre-specified gold standard classes. For a cluster-by-cluster analysis, we compute the F-measure [START_REF] Van Rijsbergen | Information Retrieval[END_REF]. To compare two partitions, we look for the best representation of the cluster a in the first partition by a cluster b in the second partition, i.e., we look for the best match between the clusters of two given partitions. This gives us as many values as there are clusters in the first partition.

The F-measure combines in a single value the measures of precision P and recall R between an a priori partition containing C clusters and the partition reached by the clustering method containing Q clusters . Let V = {v 1 , . . . , v c , . . . , v C } be the a priori partition and U = {u 1 , . . . , u q , . . . , u Q } the partition reached by the clustering method. Their contingency table is depicted in figure 5. The F-measure is then defined as follows:

F = C i=1 n i n max j=1,...,Q F (i, j), where (1)
F (i, j) = 2P (i, j)R(i, j) P (i, j) + R(i, j)

In this formula, n i indicates the number of elements in the cluster v i and n is the total number of elements.

For a global analysis, we apply the corrected Rand (CR) index [START_REF] Hubert | Comparing partitions[END_REF] to compare two partitions. The CR index also assesses the degree of agreement between an a priori partition and a partition furnished by the clustering algorithm. We use the CR index because it is not sensitive to the number of clusters in the partitions or to the distributions of the individuals in the clusters. The CR is defined as:

CR = Q i=1 C j=1 nij 2 -n 2 -1 Q i=1 ni. 2 C j=1 n.j 2 1 2 [ Q i=1 ni. 2 + C j=1 n.j 2 ] -n 2 -1 Q i=1 ni. 2 C j=1 n.j 2 (2)
where n 2 = n(n-1)

2

, n ij represents the number of objects that are in clusters u i and v j , n i. indicates the number of objects in cluster u i , n .j indicates the number of objects in cluster v j and n is the total number of objects in the data set.

The F-measure takes a value in the range [0,+1], whereas the corrected Rand index values are in the range [-1,+1]. In both cases, the value 1 indicates a perfect agreement and values near 0 correspond to cluster agreements found by chance. In fact, an analysis made by [START_REF] Milligan | A study of the comparability of external criteria for hierarchical cluster analysis[END_REF] confirmed corrected Rand index values near 0 when presented to clusters generated from random data, and showed that values lower than 0.05 indicate clusters achieved by chance.

Results

Figure 6 depicts the CR values obtained from the confrontation of the resulting partitions reached by the four different clustering strategies presented in section 2. For the F-measure, figure 7 presents boxplots summarizing the 10 values obtained (one value per cluster) on each month analysed. The partitions compared are marked in red in figures 1, 2, 3 and 4.

In figure 6, the values of the CR index reveal that the results from the local independent clustering are very different from those of the local dependent and global clustering (low values of CR means that the partitions compared are divergent). In other words, we can see by the confrontation of the local independent clustering versus the local dependent and global clustering that there are almost always low values, i.e., the local independent clustering is able to find certain clusters that were not detected by the other two clustering strategies. These conclusions are also confirmed by the F-measure (cf. figure 7).

We can also notice that the local previous clustering does not give very different results from those obtained by the local dependent clustering. The same conclusion is also valid for the confrontation between the local dependent clustering and global clustering.

Using a cluster-by-cluster confrontation via the F-measure, we refine the analysis (see figure 7). What appears quite clearly is that the clusters are very stable over time if we apply either the local previous, local dependent or global clustering strategies. In fact, no value is lower than 0.877, which represents a very good score. On the other hand, in the case of local independent clustering, we detect clusters that are very different from those obtained by the global and local dependent clustering (some values are lower than 0.5).

What is surprising is that partitions obtained by the local dependent clustering are very similar to those obtained from the global clustering. We could thus speculate whether an analysis carried out on time sub-periods would be able to obtain results supposed to be revealed by a global analysis on the entire data set.

To summarize, we can say that the local dependent clustering method shows that the clusters obtained change very little or do not change at all, whereas the local independent clustering method is more sensitive to changes which occur from one time sub-period to another.

Conclusion

In the present work, we addressed the problem of processing dynamic data in the WUM domain. The issues discussed highlight the need to define or adapt methods to extract knowledge and to follow the evolution of this kind of data. Although many powerful knowledge discovery methods have been proposed for WUM, very little work has been devoted to handling problems related to data that can evolve over time.

In this article, we have proposed a divide and conquer based approach in the sense that we split the original data set into temporal windows regarding the position of the data in the time scale. Our experiments have shown that the analysis of dynamic data by independent time sub-periods offers a certain Analysing changes in clusters of usage data over time can provide important clues about the changing nature of how a website is used, as well as the changing loyalties of its users. Furthermore, as our approach splits the data and concentrates the analysis on fewer sub-sets, some constraints regarding hardware limitations could be overcome.

Possible future work could involve the application of other clustering algorithms and the implementation of techniques that enable the automatic discovery of the number of clusters as well as identifying cluster fusions and splits over time.

Fig. 1 .

 1 Fig. 1. Global clustering

Fig. 3 .

 3 Fig. 3. Previous local clustering

Fig. 4 .

 4 Fig. 4. Dependent local clustering

Fig. 5 .

 5 Fig. 5. Contingency table

Fig. 6 .

 6 Fig. 6. Corrected Rand index values computed partition by partition

Fig. 7 .

 7 Fig. 7. F-measure values computed cluster by cluster

Table 1 .

 1 Description of the variables describing navigations

	N o Field	Signification
	1 IDNavigation	Navigation code
	2 NbRequests OK Number of successful requests (status = 200) in the navigation
	3 NbRequests BAD Number of failed requests (status = 200) in the navigation
	4 PRequests OK	Percentage of successful requests ( = NbRequests OK/ NbRequests)
	5 NbRepetitions	Number of repeated requests in the navigation
	6 PRepetitions	Percentage of repetitions ( = NbRepetitions / NbRequests)
	7 TotalDuration	Total duration of the navigation (in seconds)
	8 AvDuration	Average of duration ( = TotalDuration / NbRequests)
	9 AvDuration OK Average of duration among successful requests
		( = TotalDuration OK/NbRequests OK)
	10 NbRequests SEM Number of requests related to pages in the site's semantic structure
	11 PRequests SEM Percentage of requests related to pages in the site's semantic structure
		(=NbRequests Sem/ NbRequests)
	12 TotalSize	Total size of transferred bytes in the navigation
	13 AvTotalSize	Average of transferred bytes ( = TotalSize / NbRequests OK)
	14 MaxDuration OK Duration of the longest request in the navigation (in seconds)

This website is available at the following address: http://www.cin.ufpe.br/

Acknowledgements

The authors are grateful to CAPES (Brazil) and the collaboration project between INRIA (France) and FACEPE (Brazil) for their support for this research.


J A Silva Medeiros
email: jmedeiros@fmed.uc.pt

T M F O Gonçalves

MD Lyudmila Boyanova

M I Correia Pereira

J N Silva

Paiva De Carvalho

A M Sousa Pereira

A M Silvério Cabrita

José Augusto Da

MD, PhD Silva Medeiros

Teresa Maria Fonseca

MSc Oliveira Gonçalves

MD Marta Isabel De Correia Pereira

José Nuno Da

MSc Silva Paiva De Carvalho

MSD Anabela Maria De Sousa Pereira

António Manuel

MD, PhD Silvério Cabrita

Prof Assoc José

Augusto Da

Evaluation of eradication by triple therapy plus compared to triple therapy alone

Keywords: Helicobacter pylori, Lactobacillus acidophilus, triple therapy, eradication, urea breath test, clarithromycin, amoxicillin

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

The named authors have seen and agreed to the submitted version of the paper. The content is original and has not been published elsewhere, and the manuscript is not currently in submission for publication elsewhere.

If the contribution is accepted for publication, the paper will not be published elsewhere in the same form, in English or in any other language, without written consent of the copyright holder.

We are looking forward to receiving your answer.

With kind regards, Jose Augusto da Silva Medeiros, MD, PhD

INTRODUCTION

H. pylori infection is associated with chronic gastritis and peptic ulcer disease, and is a risk factor for the development of gastric cancer and MALT lymphoma. 1 Successful eradication of the infection by triple associations of antibiotics and proton-pump inhibitors is curative. However, the treatment may fail in 10-35% of all cases due to H. pylori resistance, antibiotic side effects and other reasons [START_REF] Megraud | H pylori antibiotic resistance: prevalence, importance, and advances in testing[END_REF]. Searching for new or additional therapeutic agents is necessary to overcome treatment failures.

There is an ongoing controversy in the literature regarding the usefulness of the addition of probiotics, including Lactobacillus acidophilus, to the standard triple therapy for H. pylori eradication [START_REF] Jm | The role of probiotics in the treatment and prevention of Helicobacter pylori infection[END_REF][START_REF] Sgouras | In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota[END_REF][START_REF] Ushiyama | Lactobacillus gasseri OLL2716 as a probiotic in clarithromycin-resistant Helicobacter pylori infection[END_REF][START_REF] Canducci | A lyophilized and inactivated culture of Lactobacillus acidophilus increases Helicobacter pylori eradication rates[END_REF][START_REF] De Francesco | Lactobacillus acidophilus administration added to omeprazole/amoxycillin-based double therapy in Helicobacter pylori eradication[END_REF][START_REF] Ziemniak | Efficacy of Helicobacter pylori eradication taking into account its resistance to antibiotics[END_REF]. While the activity of L. acidophilus , L. johnsonii and L. casei subsp. rhamnosus strains against H. pylori has been demonstrated in vitro, the in vivo successful H. pylori eradication by lactobacilli has been uncommon [START_REF] Jm | The role of probiotics in the treatment and prevention of Helicobacter pylori infection[END_REF][START_REF] Canducci | Probiotics and Helicobacter pylori eradication[END_REF][START_REF] Sakamoto | Suppressive effect of Lactobacillus gasseri OLL 2716 (LG21) on Helicobacter pylori infection in humans[END_REF].

There are relatively few studies using triple regimens supplemented with L. acidophilus in humans [START_REF] Canducci | A lyophilized and inactivated culture of Lactobacillus acidophilus increases Helicobacter pylori eradication rates[END_REF][START_REF] De Francesco | Lactobacillus acidophilus administration added to omeprazole/amoxycillin-based double therapy in Helicobacter pylori eradication[END_REF][START_REF] Ziemniak | Efficacy of Helicobacter pylori eradication taking into account its resistance to antibiotics[END_REF]. The aim of the study was to evaluate the influence of adding L. acidophilus to a triple regimen for H. pylori eradication in untreated patients with peptic ulcers or ulcer scars.

PATIENTS AND METHODS

Patients. Eligibility criteria: previously untreated H. pylori-positive adults aged 20 years or over who had endoscopically confirmed peptic ulcers or peptic ulcer scars. Exclusion criteria: previous or current treatment with proton pump inhibitors or triple therapy, and neoplastic ulcers. Design. Sixty-two consecutive H. pylori-positive patients with peptic ulcers or peptic ulcer scars, undergoing upper gastrointestinal endoscopy in an outpatient clinic in Coimbra, Portugal, were enrolled in the study. Patient characteristics are summarized in Table 1. Before treatment of the H. pylori infection, eight gastric biopsy specimens were obtained per patient: two specimens from the body and two from the antrum were used for direct Gram staining, and the remaining two specimens from the body and two from the antrum were used for culture. The patients were considered as H. Pylori-positive only if the culture was positive [START_REF] Van Leerdam | Lack of accuracy of the noninvasive Helicobacter pylori stool antigen test in patients with gastroduodenal ulcer bleeding[END_REF].

Culture and antibacterial susceptibility testing. The biopsies were frozen in Brucella broth with 25% glycerol and kept at -80º C before being cultured, as previously described [START_REF] Monteiro | Evaluation of performances of three DNA enzyme immunoassays for detection of Helicobacter pylori PCR products from biopsy specimens[END_REF]. Immediately prior to culture, the biopsies were chopped and spread evenly onto H. pylori selective medium (PYL agar, Bio Mérieux, France). The plates were incubated for 3-10 days at 37º C under microaerophilic conditions (CampyGen system, Oxoid, UK).

The suspected colonies were tested with a home-made rapid urease test (personal communication, L Monteiro, Lisboa, Portugal) and subcultured onto H. pylori selective medium (Wilkins-Chalgren agar with 10% horse blood, vancomycin 10 mg/L, cefsulodin 5 mg/L, trimethoprim 5 mg/L, and cycloheximide 100 mg/L, Biogerm, Porto, Portugal). The plates were incubated at 37° C for 48 h in a microaerophilic atmosphere (CampyGen). H. pylori identification was made by Gram staining and testing for the presence of oxidase, catalase, and urease activity.

The isolated H. pylori strains were subcultured. The minimal inhibitory concentrations (MICs) of clarithromycin and amoxicillin were evaluated against 31 strains from the patients in the EACL group and 28 strains from those in the EAC group. Three strains from the EAC group were lost while being subcultured. The strain susceptibility to the antibiotics used in this study was determined by E test, according to the method previously described [START_REF] Monteiro | Evaluation of performances of three DNA enzyme immunoassays for detection of Helicobacter pylori PCR products from biopsy specimens[END_REF][START_REF] Megraud | Antimicrobial susceptibility testing of Helicobacter pylori in a large multicenter trial: the MACH 2 study[END_REF].

The plates were incubated for 48 h in a microaerophilic atmosphere and were read according to the supplier's recommendations.

Intervention intended.

A pre-randomization design was used [START_REF] Huibers | An alternative trial design to overcome validity and recruitment problems in primary care research[END_REF]. To prevent a selection bias, the first 31 patients were allocated to therapy with esomeprazole 20 mg, amoxicillin 1000 mg and clarithromycin 500 mg, all b.i.d. for eight days (EAC group). The remaining 31 patients were allocated to therapy with esomeprazole, amoxicillin and clarithromycin (as above) plus L. acidophilus, 5x10 9 bacteria per capsule, 3 capsules in the morning and 2 capsules at night, for the same period of time (EACL group), (Table 1). The L. acidophilus probiotic used was "Lacteol" (BioSaúde laboratories, Portugal). Given the nature of the study, the patients were not blinded to the assigned treatment.

Urea breath test. To detect possible differences in H. pylori eradication rates between the patients of EACL group and those of EAC group, a 13 C urea breath test (UBT, Helico-Test 13 C-Ureia, Isomed S.L. Pharmaceutical Laboratory, Madrid, Spain) was performed six to seven weeks after the end of therapy. The specimen collection was performed immediately before (basal sample) and 30 min after (post-test sample) the ingestion of the marked urea; the 13 C/ 12 C ratio was measured in both specimens, and the result was expressed as the difference between the basal and post-test measurements. The UBT was considered positive when this value was >5 delta over baseline (DOB) per 1,000. This study had a single-blind design, with the technicians administering the UBT and evaluating the outcomes being blinded to patient group assignments.

Statistical analysis.

The chi-square test and Fisher's exact test of independence (when appropriate) were used for the statistical analysis. The outcome variable was the presence of H. pylori eradication (yes vs no) and the exposure variable was Lactobacillus supplementation of the triple therapy (yes vs no). A p value of <0.05 was considered statistically significant.

This study was approved by the Ethics Committee of the Social Services of the University of Coimbra (Coimbra, Portugal) with the registration number 36/07. It was registered at ClinicalTrials.gov, of the National Institutes of Health (USA), with the protocol ID "SASUC-36/07". Informed written consent was obtained from all patients.

RESULTS

The MICs of the 31 strains in the EACL group and the 28 strains in the EAC group for amoxicillin were MIC 50 , <0.016 and <0.016 mg/L, MIC 90 , 0.023 and 0.023 mg/L, and ranges of <0.016-0.047 and <0.016-0.19 mg/L, respectively. The MICs of the 31 strains in the EACL group and the 28 strains in the EAC group for clarithromycin were MIC 50 , <0.016 and <0.016 mg/L, MIC 90 , 0.032 and 0.064 mg/L, and ranges of <0.016->256 and <0.016-48 mg/L, respectively.

Successful eradication rates, as determined by a negative UBT after treatment, were similar in the patients of EAC group (80.6%, 25 of 31) and in those of EACL group (83.9%, 26 of 31, p=0.740, OR 0.80; 95% CI: 0.22-2.96) by both intention to treat and per protocol analysis. The strains of the 11 non-eradicated patients were in vitro susceptible to both clarithromycin and amoxicillin. The MIC ranges against the five strains of EACL group and the six strains of EAC group were <0.016-0.016 and <0.016-0.19 mg/L for clarithromycin, and <0.016-0.047 mg/L and <0.016-0.19 for amoxicillin, respectively. Safety. There were no reported dropouts from the study due to non-compliance of the patients or side effects from the medications.

DISCUSSION:

Probiotics are live, nonpathogenic microbial feeds or food supplements that have been used to improve the balance of the microflora, primarily in the gastrointestinal tract [START_REF] Franceschi | Role of probiotics in patients with Helicobacter pylori infection[END_REF].

Lactobacilli are acid resistant and can persist in the stomach longer than most other bacteria [START_REF] Bhatia | Lactobacillus acidophilus inhibits growth of Campylobacter pylori in vitro[END_REF]; therefore, they have the potential to inhibit H. pylori, both for prophylaxis and for treatment of the infection.

Previous studies have found that the supplementation of standard anti-H. pylori regimens with probiotics can reduce the side effects of triple therapy and, therefore, can improve patients' compliance to therapy [START_REF] Franceschi | Role of probiotics in patients with Helicobacter pylori infection[END_REF].

Other authors have identified the in vitro activity of L. acidophilus against H. pylori, which has been associated with the secretion of lactic acid, CRL639 autolysins and competition for adhesion [START_REF] Lesbros-Pantoflickova | Helicobacter pylori and probiotics[END_REF]. However, most of these properties have been observed only in animal models or in vitro data. In addition, the activity of several probiotic species against H. pylori has been reported to be strain-dependant [START_REF] Kim | Antagonism of Helicobacter pylori by bacteriocins of lactic acid bacteria[END_REF].

In vivo, the administration of probiotics alone usually does not lead to H. pylori eradication [START_REF] Lesbros-Pantoflickova | Helicobacter pylori and probiotics[END_REF]. However, in several studies, adding Lactobacillus species (e.g., L. johnsonii La1 lyophilized + inactivated culture or L. casei DN 114001) to a triple therapy of amoxicillin, clarithromycin and a proton pump inhibitor has increased the eradication rate compared to the standard triple regimen alone [START_REF] Franceschi | Role of probiotics in patients with Helicobacter pylori infection[END_REF].

In the present study, the addition of 1.5x10 10 L. acidophilus organisms in the morning and 1x10 10 organisms at night, failed to improve H. pylori eradication. Although 13 C UBT was performed at least six weeks after the end of therapy, the annual recurrence of H. pylori infection has been reported to be low (<1-2.7%) in most developed countries [START_REF] Niv | H pylori recurrence after successful eradication[END_REF]; therefore, UBT-positivity in our sample is most likely due to persistent infection than to reinfection, the latter being unlikely to influence the study outcome.

There are several possible reasons for the absence of effect observed in this study.

Firstly, the choice of probiotic to be added to the standard therapy seems to be important. It is still unclear which Lactobacillus species and strains are most active against H. pylori. So far, the most effective (both in vitro and in vivo) Lactobacillus species have been L. casei and L. johnsonii La1 [START_REF] Lesbros-Pantoflickova | Helicobacter pylori and probiotics[END_REF]. In the relatively few studies on the addition of L. acidophilus to the standard eradication regimens, only two sets of data have reported an increase in H. pylori eradication rate. However, in one of the successful studies, a probiotic preparation containing both L. acidophilus and L. rhamnosus had been used [START_REF] Canducci | A lyophilized and inactivated culture of Lactobacillus acidophilus increases Helicobacter pylori eradication rates[END_REF][START_REF] Ziemniak | Efficacy of Helicobacter pylori eradication taking into account its resistance to antibiotics[END_REF].

In addition, the length (14 days in the study by De Francesco et al. [START_REF] De Francesco | Lactobacillus acidophilus administration added to omeprazole/amoxycillin-based double therapy in Helicobacter pylori eradication[END_REF], three weeks in the study by Cremonini et al. [START_REF] Cremonini | Effect of different probiotic preparations on anti-Helicobacter pylori therapy-related side effects: a parallel group, triple blind, placebo-controlled study[END_REF] and 8 days in the present study) and frequency of administration (t.i.d. in the study of Canducci et al. 5 and b.i.d. in the present study) could also be of importance.

Finally, a combination of probiotics could provide a better effect on H. pylori eradication than the use of single species. Several authors [START_REF] Ziemniak | Efficacy of Helicobacter pylori eradication taking into account its resistance to antibiotics[END_REF][START_REF] Sheu | Impact of supplement with Lactobacillus-and Bifidobacterium-containing yogurt on triple therapy for Helicobacter pylori eradication[END_REF] have obtained an increase in H. pylori eradication, compared to the standard therapy with probiotic combinations such as Lactobacillus-and Bifidobacterium-containing yogurt (AB-Yogurt) or L. acidophilus and L. rhamnosus. A recent study has demonstrated a significantly higher H. pylori eradication rate by a quadruple therapy following pre-treatment with AB-yogurt containing a mixture of L. acidophilus La5, Bifidobacterium lactis Bb12, Lactobacillus bulgaricus, and Streptococcus thermophilus, than by the quadruple therapy alone [START_REF] Sheu | Pretreatment with Lactobacillus-and Bifidobacterium-containing yogurt can improve the efficacy of[END_REF].

CONCLUSION

H. pylori eradication rates in the EAC and EACL groups were comparable: adding L. acidophilus to the triple therapy used in this study did not increase the eradication rate.

Considering both the patients' expenses and the need for the daily ingestion of five extra capsules, the supplementation of the amoxicillin + clarithromycin + esomeprazole therapy with L. acidophilus, at a daily dose of 2.5 x 10 10 cells, does not benefit the ulcer patient.

Further studies with different posologies or duration of treatment, and with alternative probiotics or probiotic combinations are required to improve the eradication of H. pylori infection.

Conflict of interest statement:

The authors declare that they have no conflicts of interest, including financial or personal relationships with other people or organisations, that could influence their work. quadruple therapy in eradicating residual Helicobacter pylori infection after failed triple therapy. Am J Clin Nutr 83:864-869.

  the manuscript titled "Evaluation of Helicobacter pylori eradication by triple therapy plus Lactobacillus acidophilus compared to triple therapy alone" by Medeiros JAS et al. for consideration for publication in the European Journal of Clinical Microbiology & Infectious Diseases.

Table 1 .

 1 Patients involved in the study.

	Group	Parameter	Patients in the EACL	Patients in the
			group	EAC group
	Sex	Men	18 (58.1%)	14 (45.2%)
		Women	13 (41.9%)	17 (54.8%)
	Age	Average (years)	50.7	53.8
		Median (years)	52.0	58.5
		Range (years)	22-80	24-78
	Diseases	Duodenal ulcer	20 (64.5%)	23 (74.2%)
		Duodenal ulcer scar	10 (32.3%)	5 (16.1%)
		Gastric ulcer	1 (3.2%)	3 (9.7)
	Total	All patients	31	31
	Abbreviations: EAC group: patients treated by the standard triple therapy -esomeprazole 20
	mg, amoxicillin 1000 mg and clarithromycin 500 mg, all b.i.d. for 8 days; EACL group:
	patients treated by the triple therapy plus L. acidophilus -5x10 9 organisms per capsule, 3
	capsules in the morning and 2 capsules at night, for the same period of time.

Table 2 .

 2 H. pylori eradication by adding Lactobacillus acidophilus to the eradication regimens of patients with gastroduodenal diseases.

	No. of	No. of	L.	Duratio	Regime	Eradicatio	Eradication	Significa	Referenc
	patients	patient	acidophil	n of	n	n rate (%)	rate (%) by	nt	e
	treated	s	us	treatme		by classic	probiotic-	increase
	by	treated		nt		regimen	supplement	in
	standar	by		(days)			ed regimen	eradicatio
	d	standar						n rate
	regime	d
	n +	regime
	probioti	n
	c
	60	60	L.	7	RCA 70 (ITT),	87 (ITT),	Yes	5
			acidophil			72 (PP)	88 (PP)
			us
			lyophilize
			d and
			inactivate
			d culture
			(t.i.d.)
	47	37	L.	14	OA	70.3	63.8	No	6
			acidophil
			us LB
			spent
			culture
			supernata
			nt
	53	192 Lacidofil	10	PPI-AC	85.9	94.3	Yes	7
			containin
			g L.
			acidophil
			us and L.
			rhamnosu
			s (two
			capsules
			b.i.d.)
	31	31	L.	8	EAC	80.6	83.9	No	Present
			acidophil			(ITT and	(ITT and		study
			us 5x10 9			PP)	PP)
			organisms
			per
			capsule,
			(b.i.d.-3
			capsules
			in the
			morning
			and 2

Table 1 .

 1 Patients involved in the study.

	Group	Parameter	Patients in the EACL	Patients in the
			group	EAC group
	Sex	Men	18 (58.1%)	14 (45.2%)
		Women	13 (41.9%)	17 (54.8%)
	Age	Average (years)	50.7	53.8
		Median (years)	52.0	58.5
		Range (years)	22-80	24-78
	Diseases	Duodenal ulcer	20 (64.5%)	23 (74.2%)
		Duodenal ulcer scar	10 (32.3%)	5 (16.1%)
		Gastric ulcer	1 (3.2%)	3 (9.7)
	Total	All patients	31	31
	Abbreviations: EAC group: patients treated by the standard triple therapy -esomeprazole 20
	mg, amoxicillin 1000 mg and clarithromycin 500 mg, all b.i.d. for 8 days; EACL group:
	patients treated by the triple therapy plus L. acidophilus -5x10 9 organisms per capsule, 3
	capsules in the morning and 2 capsules at night, for the same period of time.

Table 2 .

 2 H. pylori eradication by adding Lactobacillus acidophilus to the eradication regimens of patients with gastroduodenal diseases. Abbreviations: ITT: intention to treat analysis., PP: per protocol analysis; RCA: rabeprazole (20 mg b.i.d.), clarithromycin (250 mg t.d.s.) and amoxicillin (500 mg t.d.s.); OA: omeprazole + amoxicillin; EAC: esomeprazole 20 mg b.i.d., amoxicillin 1000 mg b.i.d. and clarithromycin 500 mg b.i.d., for 8 days; PPI-AC: amoxicillin with clarithromycin and proton pump inhibitor.

	No. of	No. of	L.	Duratio	Regime	Eradicatio	Eradication	Significa	Referenc
	patients	patient	acidophil	n of	n	n rate (%)	rate (%) by	nt	e
	treated	s	us	treatme		by classic	probiotic-	increase
	by	treated		nt		regimen	supplement	in
	standar	by		(days)			ed regimen	eradicatio
	d	standar						n rate
	regime	d
	n +	regime
	probioti	n
	c
	60	60	L.	7	RCA 70 (ITT),	87 (ITT),	Yes	5
			acidophil			72 (PP)	88 (PP)
			us
			lyophilize
			d and
			inactivate
			d culture
			(t.i.d.)
	47	37	L.	14	OA	70.3	63.8	No	6
			acidophil
			us LB
			spent
			culture
			supernata
			nt
	53	192 Lacidofil	10	PPI-AC	85.9	94.3	Yes	7
			containin
			g L.
			acidophil
			us and L.
			rhamnosu
			s (two
			capsules
			b.i.d.)
	31	31	L.	8	EAC	80.6	83.9	No	Present
			acidophil			(ITT and	(ITT and		study
			us 5x10 9			PP)	PP)
			organisms
			per
			capsule,
			(b.i.d.-3
			capsules
			in the
			morning
			and 2

ACKNOWLEDGMENTS

The authors would like to thank the "Dr. Ricardo Jorge" National Institute of Health, Portugal, for its support in the execution of the microbiology tests.


Tania M Murphy

Alex Y Nilsson

Ipsita Roy

Anthony Harrop

Keith Dixon

Tajalli Keshavarz
email: t.keshavarz@wmin.ac.uk

Enhanced intracellular Ca concentrations in and after addition of oligosaccharide elicitors

Keywords: Oligosaccharide elicitors, intracellular calcium, Apoaequorin, secondary metabolites

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

Introduction

Elicitation using carbohydrates stimulates morphological and physiological responses in fungi and bacteria. The overproduction of a range of antibiotics and enzymes has been achieved by adding small traces of oligosaccharide elicitors to cultures [START_REF] Petruccioli | Enhancement of glucose oxidase production by Penicillium variable P16[END_REF][START_REF] Nair | Optimisation studies on multiple elicitor addition in microbial systems: P. chrysogenum and B. Licheniformis[END_REF]Murphy et al. 2007a). The effect of elicitation on fungal morphology and penicillin G production has also been studied: Penicillium chrysogenum supplemented with oligosaccharide elicitors had higher hyphal tip numbers compared to control cultures.

Enhancement of penicillin G in elicited cultures may be related to the morphological changes caused by the elicitation [START_REF] Radman | Elicitor effects on Penicillium chrysogenum morphology in submerged cultures[END_REF]). Elicitors also effect the transcription of genes for antibiotic biosynthesis (Murphy et al. 2007 b;[START_REF] Nair | Quantitative PCR study on the mode of action of oligosaccharide elicitors on penicillin G production by Penicillium chrysogenum[END_REF]. However, a possible mechanism by which elicitors cause these changes has yet to be defined.

Ca 2+ is widely recognised as a secondary messenger that transmits external information into the eukaryotic cell [START_REF] Campbell | Intracellular calcium: Its universal role as regulator, Wiley and Sons Ltd, Chichester Dominguez DC (2004) Calcium signalling in bacteria[END_REF]. Eukaryotes maintain a very low cytoplasmic Ca 2+ concentration and Ca 2+ homeostasis has been reported for many years (Knight et al. 1991 a). The role of Ca 2+ in bacteria is less clear; however, it may be involved in prokaryotic cell division, chemotaxis, motility, competence, sporulation, cell defence, synthesis of specific proteins, gene expression and stress signals [START_REF] Norris | Calcium signalling in bacteria[END_REF][START_REF] Jones | Direct measurement of free Ca 2+ shows different regulation of Ca 2+ between the periplasm and the cytosol of Escherichia coli[END_REF]Dominguez, 2004).

Cytosolic Ca 2+ concentration in E. coli is similar to that in eukaryotic systems suggesting that bacteria regulate Ca 2+ homeostasis and that the Ca 2+ gradient could be used for transmitting information inside the cell [START_REF] Jones | Direct measurement of free Ca 2+ shows different regulation of Ca 2+ between the periplasm and the cytosol of Escherichia coli[END_REF]. Due to the variety of responses observed after elicitation, we have investigated the effect of oligosaccharide elicitors on bacterial Ca 2+ homeostasis; this adds to the knowledge base for the mechanism of elicitation contributing to a more robust system for enhanced secondary metabolite production via elicitation.

Methods

All materials were purchased from Sigma, unless stated otherwise. E. coli DH5α and B. subtilis 1604 were used as models for Gram-negative and Gram-positive bacterial cultures.

Preparation of elicitors

Mannan oligosaccharide (MO) was prepared by enzymatic hydrolysis of locust bean gum; oligoguluronate (OG) and oligomannuronate (OM) were prepared by partial acid hydrolysis of sodium alginate [START_REF] Asilonu | Enhancement of chrysogenin production in cultures of Penicillium chrysogenum by uronic acid oligosaccharides[END_REF].

Elicitors were stored at 4 °C.

Plasmids

To measure the intracellular Ca 2+ in bacteria and to investigate the effect of oligosaccharide elicitors on calcium fluxes, aequorin technology was used. The apoaequorin protein from the jelly fish, Aequorea victoria, forms a complex with coelenterazine (the prosthetic group) and, upon Ca-binding, the aequorin complex emits bioluminescence allowing the measurement of intracellular Ca 2+ . The pMMB66EH (Amp r ) containing the apoaequorin cDNA was a kind gift from Prof. Anthony Campbell (Cardiff University, UK). The apoaequorin gene was inserted between the restriction sites SalI and PstI at the multiple cloning site of the pMMB66EH. To express the apoaequorin gene in E. coli and B. subtilis, an E. coli-B. subtilis shuttle vector pHCMC05 (Bacillus Genetic Stock Center, USA) was used. This vector contains an IPTG-inducible Spac promoter, which is located upstream of the multiple cloning site and also harbours chloramphenicol-and ampicillin-resistance markers.

Cloning of apoaequorin gene

On the multiple cloning site in the shuttle vector pHCMC05, XmaI and XbaI were the restriction sites chosen for cloning of the apoaequorin gene. XmaI and XbaI sites were engineered onto the 5'-and 3'-ends of the aequorin sequence during PCR reaction. The primers used were (5'-to 3'-):

GGATCCTCTAGAATGACCAGCGAACAATAC and CTGCAGCCCGGGTTAGGGGACAGCTCCACC.

The PCR products were visualised on 1% (w/v) agarose gel and were purified using Qiagen Gel Extraction kit (Qiagen, UK) following the manufacturer's instructions. For the restriction and ligation reactions, all the enzymes were purchased from New England Biolabs, UK. The pHCMC05 vector and PCR products were digested with XbaI and XmaI at 37 °C for 2 h. The digested PCR product and the plasmid were gel-purified using Qiagen Gel Extraction kit. The digested plasmid, PCR product, T4 DNA ligase buffer and T4 DNA ligase were mixed and incubated at 16 °C for 16 h. Ligation reactions were used to transform E. coli DH5α and B.

subtilis 1604 using the electrophoretic method by [START_REF] Dower | High efficiency transformation of E. coli by high-voltage electroporation[END_REF] and [START_REF] Xue | High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis[END_REF], respectively.

Plasmid preparation was carried out using QIAprep spin miniprep kit (Qiagen), following the manufacturer's instructions. Sequencing of the constructed clone was carried out by GATC (Germany). The following primer sequence was designed (5'-to 3'-): CATTTGTTCCAGGTAAGG, with a melting temperature of 65 °C and a GC content of 47%.

Growth of E. coli and B. subtilis expressing apoaequorin

Five ml of bacterial cultures were grown in LB medium containing 100 µg ampicillin ml -1 or 5 µg chloramphenicol ml -1 for E. coli DH5α and B. subtilis 1604, respectively. Incubation was carried out at 30 °C at 250 rpm.

Expression of apoaequorin in bacterial cultures

0.5 ml of an overnight culture was inoculated into 10 ml LB medium containing the selective antibiotic. When the OD 600 reached 0.45 , the culture was induced with 0.4 mM IPTG for 2.5 h according to [START_REF] Nguyen | Construction of plasmidbased expression vector for Bacillus subtilis exhibiting full structural stability[END_REF].

Reconstitution of aequorin in vivo with coelenterazine H

To reconstitute aequorin, cells were centrifuged at 3000 g for 5 min and re-suspended in Buffer A (25 mM HEPES, 125 mM NaCl, 1 mM MgCl 2 , pH 7.5) containing 2.5 µM coelenterazine H (Invitrogen) followed by incubation at room temperature in dark for 1 h.

Measurement of intracellular Ca 2+ levels in bacterial system

Cells expressing functional aequorin were centrifuged and resuspended in either of LB medium (in the case of E. coli DH5α) or Buffer A (in the case of B. subtilis 1604) in the presence or absence of 0.5 mM ethylene glycol tetraacetic acid (EGTA). Cells (100 µl) were challenged with CaCl 2 and elicitors. Luminescence readings were measured with a microplate reader and the light emitted was measured as relative luminescence units (RLU).

Buffer A and LB were used to set the luminescence background. White Cornwell 96 well-plates were used for this assay.

Results

Elicitation effects on intracellular Ca 2+ levels in bacterial cultures

To investigate whether the addition of oligosaccharide elicitors changes the intracellular Ca 2+ in bacteria, the apoaequorin gene was cloned into a Gram-negative/Gram-positive shuttle vector pHCMC05. The pHCMC05-Aq vector was digested with XmaI-XbaI. The released insert was 597 bp which was the expected size (Fig. 1).

The apoaequorin gene in the donor and the expression vectors (PMMB66EH and pHCM05-Aq) were sequenced thereby confirming that no mutations had occurred during PCR and that the insert was correctly inserted in the expression shuttle vector (data not shown).

Fig. 1 Expression of apoaequorin using 0.4 mM IPTG and its reconstitution with coelenterazine H was conducted as described in Methods. E. coli DH5α without the pHCMC05-Aq plasmid was tested for luminescence in the presence or absence of Ca 2+ , elicitors, water and the luminescence exhibited by the culture was zero in all cases (data not shown). E. coli DH5α pHCM05-Aq cultures expressing aequorin were challenged with 100 mM CaCl 2 followed by elicitors. The elicitors were added at concentrations previously chosen to enhance secondary metabolites in bacteria (Murphy et al. 2007 a). Supplementation of E. coli DH5α pHCM05-Aq with oligosaccharide elicitors (MO at 300 mg l -1 ; OM at 200 mg l -1 or OG at 100 mg l -1 ), after the first 100 mM CaCl 2 addition, increased Ca 2+ levels (Fig. 2) by 11-fold with OG and by 7-and 5-fold with MO and OM, respectively.

Fig. 2

We then investigated the effect of Ca 2+ and elicitors in B. subtilis 1604 pHCM05-Aq using identical conditions to those used for E. coli DH5α pHCM05-Aq experiments. As no luminescence signal was detected under those conditions, lower CaCl 2 concentrations were tested without success (data not shown). When B. subtilis 1604 pHCM05-Aq cells were resuspended in Buffer A (+EGTA) rather than LB broth (Fig. 3a), addition of 0.1 mM CaCl 2 increased the intracellular Ca 2+ levels (Fig. 3b). We therefore interrogated the influence of elicitors in the absence of extracellular Ca 2+ in B. subtilis 1604 pHCM05-Aq. Individual addition of OG or MO increased Ca 2+ levels by 168-and 33-fold, respectively.

Addition of water caused an increase of 4-fold. When cells were challenged with OG followed by MO, 170-and 39-fold increases in cytosolic Ca 2+ levels were observed (Fig 5).

Fig. 5

Discussion

Calcium signalling has recently been recognised to be present in bacterial cultures. The role of Ca 2+ has been harder to demonstrate due to the difficulties in measuring intracellular Ca 2+ levels in this type of cells. This is due to the size of the cells, the presence of cell wall, dye loading into the bacteria, dye toxicity and background fluorescence [START_REF] Norris | Calcium signalling in bacteria[END_REF]. Aequorin technology however, can measure intracellular Ca 2+ levels in living cells without any of the above mentioned drawbacks [START_REF] Norris | Calcium signalling in bacteria[END_REF].

Calcium homeostasis, calcium channels, calcium-binding proteins and a calcium-dependent adenylate cyclise (Knight et al. 1991 b) have been reported in E. coli and B. subtilis [START_REF] Herbaud | Calcium signalling in Bacillus subtilis[END_REF][START_REF] Jones | Direct measurement of free Ca 2+ shows different regulation of Ca 2+ between the periplasm and the cytosol of Escherichia coli[END_REF]Dominguez, 2004). Alterations in intracellular Ca 2+ levels trigger cell activation through protein phosphorylation [START_REF] Campbell | Intracellular calcium: Its universal role as regulator, Wiley and Sons Ltd, Chichester Dominguez DC (2004) Calcium signalling in bacteria[END_REF][START_REF] Norris | Calcium signalling in bacteria[END_REF].

In this work, the effect of exogenous calcium was tested in E. coli pHCM05-Aq and B. subtilis pHCM05-Aq cultures. Ca 2+ transients were observed upon CaCl 2 addition with resting levels reestablishing rapidly as reported by other investigators (Knight et al. 1991-b;[START_REF] Jones | Slow changes in cytosolic free Ca 2+ in E. coli highlight two possible influx mechanisms in response to changes in extracellular calcium[END_REF][START_REF] Watkins | Free calcium transients in chemotactic and nonchemotactic strains of Escherichia coli determined by using recombinant aequorin[END_REF][START_REF] Herbaud | Calcium signalling in Bacillus subtilis[END_REF]. Transient response in Ca 2+ levels due to external stimulus should disappear fast for the cell to generate a rapid and efficient response to new situations [START_REF] Norris | Calcium signalling in bacteria[END_REF].

As shown in Fig. 2 and 3, higher exogenous calcium concentrations were required to create a notable intracellular Ca 2+ increase in E. coli compared to B. subtilis cultures. Similar observations were reported in bacterial cultures during the investigation of the role of Ca 2+ in chemotaxis. Higher external calcium levels were required by E. coli (1 mM) compared to B. subtilis (100 nM) to promote Ca 2+ transients that would result in the cells tumbling instead of swimming. The differences in exogenous calcium concentration required by both cultures to generate intracellular Ca 2+ changes are thought to be due to the difference in the cell envelope/wall structures between Gram-positive and Gram-negative bacteria [START_REF] Watkins | Free calcium transients in chemotactic and nonchemotactic strains of Escherichia coli determined by using recombinant aequorin[END_REF].

Different Ca 2+ signatures were observed upon addition of OG or MO to E. coli pHCM05-Aq and B. subtilis pHCM05-Aq, suggesting that different calcium messages might be transmitted probably due to the difference in the three-dimensional structure between MO and OG. This may explain why higher antibiotic levels are achieved when Penicillium chrysogenum and Bacillus licheniformis are stimulated with multiple elicitor additions using different elicitors (OG and MO) in comparison to repeated addition of the same elicitor to the culture (Murphy et al. 2007-b;[START_REF] Nair | Quantitative PCR study on the mode of action of oligosaccharide elicitors on penicillin G production by Penicillium chrysogenum[END_REF].

Addition of different elicitors to Nicotiana plumbaginifolia produced different Ca 2+ signatures varying in intensity and duration. The increase in intracellular Ca 2+ levels is a fast event in elicitor sensing mechanisms in plants and is dependent on the interaction and binding of elicitors to the specific receptors [START_REF] Lecourieux | Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells[END_REF]. Similar results were obtained by Knight et al. (1991-a) studying the effect of yeast elicitors on Nicotiana plumbaginifolia. The authors suggested that signalling occurs through Ca 2+ level changes and the signal transmitted depends on the Ca 2+ signatures.

The results presented in this study confirm that E. coli and B. subtilis cultures are able to regulate intracellular Ca 2+ levels and to sense external signals which are transmitted through Ca 2+ transients. Here we report, for the first time, the effect of elicitors on intracellular Ca 2+ in bacterial cultures. It is suggested that cytosolic Ca 2+ levels increase due to addition of OG and MO elicitors and therefore the effects on secondary metabolite production and gene expression observed in many fungal and bacterial cultures upon addition of elicitors is, at least partly, due to the increase in Ca 2+ levels which activates the signalling mechanisms in both systems.

Further investigation into the elucidation of elicitation mechanism and its understanding could lead to the application of this novel, cheap and easy method for enhancement of secondary metabolites production at largescale. at 300 mg l -1 ) followed by water; (b) water followed by mannan oligosaccharide (MO at 300 mg l -1 ); (c) oligoguluronate (OG at 100 mg l -1 ) followed by water; (d) oligoguluronate (OG at 100 mg l -1 ) followed by mannan oligosaccharide (MO at 300 mg l -1 ). Cells were re-suspended in buffer A (in the presence of EGTA).

Arrows represent the addition of either water or elicitors to the culture

Fig. 3

 3 Fig. 3 Stimulation of B. subtilis 1604 pHCM05-Aq cells with OG and MO, after CaCl 2 addition, increased Ca 2+ levels significantly (p<0.05) by 10-and 3-fold, respectively. Neither water nor OM, changed the Ca 2+ levels as shown in Fig. 4.Fig. 4

Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5

 12345 Fig. 1 Screening of positive colonies. Restriction digest of pHCMC05-Aq from E. coli DH5α. Lane M: 2-log DNA ladder (New England Biolabs). Arrows show the position of the released insert. Undigested pHCMC05-Aq: lanes 1, 3, 5. Double digest of pHCMC05-Aq with XmaI-XbaI: lanes 2, 4, 6

  Fig. 1

Acknowledgements

We would like to thank Prof. Anthony Campbell (University of Cardiff, UK) for his guidance and Pfizer Ltd for Tania Murphy's studentship.