Alison R Evans

Catherine H Mercer

Violetta Parutis

Graham J Hart

Richard Mole

Christopher J Gerry

Dr Fiona M Burns
email: f.burns@ucl.ac.uk

Factors associated with GUM clinic attendance and STI diagnosis among Central and East European migrants in London

Keywords: GUM Services, Health Service Research, Sexual Behaviour, Sexual Health, STD, European migration, UK

Objectives: There has been a large influx of Central and East European (CEE) migrants to the UK following the expansion of the European Union. This paper examines the factors associated with GUM clinic attendance and STI diagnosis among CEE migrants in London.

Methods:

We conducted a survey of sexual behaviour among CEE migrants attending two central London GUM clinics (n=299) and community venues in London (n=2276). Routinelycollected clinic data were also analysed.

Results: CEE migrants made up 2.9% of male and 7.0% of female attendees at the clinics. Half of the women attending sessions for female sex workers were from CEE countries and paying for sex was widely reported by men. Women were more likely than men to have attended a GUM clinic in the UK (7.6% vs 4.5%, p=0.002). GUM survey respondents were more likely than community survey respondents to report one or more new sexual partners in the past year (women: 67.9% vs 28.3%, p<0.001; men: 75.6% vs 45.1%, p<0.001) and homosexual partnership(s) in the past five years (men: 54.3% vs 1.8%, p<0.001); but were less likely to report assortative heterosexual mixing (women: 25.9% vs 74.2%, p<0.001; men: 56.5% vs 76.3%, p<0.001).

Conclusions: CEE patients make up a notable minority of patients attending two central London GUM clinics. Higher numbers of sexual partners, homosexual partnerships and sexual mixing with people from outside the country of origin are associated with GUM clinic attendance.

Heterosexual CEE men report behaviours associated with HIV/STI acquisition but appear to be under-utilizing GUM services.

Key messages

• The accession of ten Central and East European (CEE) countries has resulted in a large influx of predominantly young economic migrants to the UK.

• Attendances at central London GUM services by migrants from eight of these accession countries have increased steadily over a number of years.

• CEE GUM clinic attendance is associated with reporting new sexual partners, homosexual partnerships and heterosexual mixing with people from outside the home country.

• CEE migrants make up a notable minority of patients at central London GUM services but these services may be under-utilized by heterosexual CEE men.

INTRODUCTION

On 1 st May 2004, the Czech Republic, Estonia, Hungary, Latvia, Lithuania, Poland, Slovakia and Slovenia (Accession 8 or A8) joined the European Union (EU), followed by Romania and Bulgaria (A2) on 1 st January 2007. The accession of these ten Central and East European (CEE) countries resulted in a large influx of predominantly young economic migrants to the UK. All nationals of these countries have entitlement to NHS services and the UK has a responsibility to ensure that the health needs of these communities are met.

In a recent study of people who attended two central London GUM clinics, 2.2% of men and 6.6% of women were from an A8 country and the proportion of attendances by A8 men and women had increased significantly over six years. [START_REF] Burns | Increased attendances by people of Eastern European origin at sexual health services in London[END_REF] However, these data do not provide any information on sexual risk behaviour which would enhance our understanding of the sexual health service needs of people from CEE countries. While reporting GUM clinic attendance is associated with greater numbers of sexual partners, having same-sex partnerships, injecting drug use and paying for sex among those in the general British population, [START_REF] Fenton | Reported sexually transmitted disease clinic attendance and sexually transmitted infections in Britain: prevalence, risk factors, and proportionate population burden[END_REF] the factors lying behind GUM clinic attendance among the new CEE migrant population are unknown. Following the accession of the A2 countries, this paper examines sexual risk factors among men and women from both the A8 and A2 countries who attend sexual health services in London, as part of the SALLEE project (Sexual Attitudes and Lifestyles of London's East Europeans).

METHODS SALLEE cross sectional survey

A detailed description of the methodology has been published. [START_REF] Evans | The sexual attitudes and lifestyles of London's Eastern Europeans (SALLEE Project): design and methods[END_REF] A summary of the methods for the SALLEE cross sectional survey is described below. In this paper we refer to two samples of men and women who took part in the survey -SALLEE GUM respondents and SALLEE community respondents.

Participants and procedure

Eligible respondents for both samples were literate men and women aged 18 years or over who self-identified as migrants from one of the ten CEE countries. Eligible respondents were born or spent formative years in one of these countries. Given the fluid nature of migration, length of time in the UK was not a pre-condition for participation. The SALLEE GUM sample was recruited from the two central London GUM clinics over nine months (1 st

Sampling

For the SALLEE GUM sample, the days and times of recruitment in the clinics varied over the data collection period. During the times that recruitment took place, men and women who stated on their clinic registration form that they had been born in one of the ten CEE countries were asked to participate in the study. For the SALLEE community sample, recruitment locations were selected following a detailed social mapping exercise and times of recruitment at venues were varied. Fieldworkers asked individuals where they were from before asking if they would be willing to take part in the study. The resulting community sample reflects a broad crosssection of CEE migrants in London.

Study instrument

The survey instrument was a self-completed questionnaire that was fielded using hand-held computers and included no information that would allow respondents to be identified. The questionnaire was available in twelve languages (the ten CEE languages plus English and Russian). It was translated from English into the eleven languages and checked for accuracy by native speakers of these languages. Informed consent was sought using information sheets available in the twelve languages. A £5 high street voucher was offered as an incentive.

The questionnaire concentrated on sexual risk behaviour, including sexual practices, numbers of partnerships, use of condoms, paying for sex, STIs (including HIV) and use of sexual health services. Respondents were also asked about recreational drug use. Where possible, questions from previously validated questionnaires were used in order to maximize their reliability and validity. The questionnaire was piloted with nine CEE migrants to examine its feasibility and acceptability and to explore understanding of the question items and underlying constructs. The final questionnaire took about ten minutes to complete.

Retrospective analysis of routinely-collected GUM clinic data

Routinely-collected data on all attendances of patients from CEE countries and elsewhere at the same two GUM clinics were analysed for the same period (1 st July 2008 to 31 st March 2009).

We refer to these data as GUM patients in order to distinguish them from the SALLEE GUM respondents who participated in the cross sectional survey. Data analysed here refer to new attendances; GUM attendees who were known to be HIV positive prior to their visit were excluded, as were follow-up GUM appointments unless they resulted in a new STI diagnosis.

Statistical analysis

RESULTS

Retrospective analysis of routinely-collected GUM clinic data

This analysis of routinely-collected GUM clinic data was conducted for the same period as data collection for the SALLEE project (1 st July 2008 to 31 st March 2009) and provides the context for our analysis of the SALLEE cross sectional survey. A total of 29,770 GUM patients attended the two clinics at least once during this time and half of them were male (48.7%). Country of origin was not recorded for 8.9% of GUM patients. Of the 27,131 men and women with a known country of origin, 1,373 (5.1%) were born in a CEE country: 2.9% of men and 7.0% of women.

One of the GUM clinics runs sessions specifically for female sex workers (FSW). Among the 295 patients attending these sessions, 50.2% (n=148) were from a CEE country, compared to 6.1% of female patients attending all other GUM sessions. The majority of CEE women attending sessions for FSWs were Romanian (44.6%), Lithuanian (25.7%) or Polish (10.8%). As the SALLEE cross sectional survey was not administered to women attending sessions for FSWs, these women are excluded from the remaining analysis of clinic data.

Overall, male GUM patients were more likely than females to have one or more STI diagnoses (including HIV) during their visits (18.5% vs 13.3%, p<0.001). There was no significant difference between men from CEE countries and from the UK or elsewhere on the overall likelihood of an STI diagnosis (20.5% vs 18.5%, p=0.338). CEE women were slightly less likely to be diagnosed with an STI than women from the UK or elsewhere (11.0% vs 13.6%, p=0.032).

A higher proportion of CEE men were recorded as homosexual or bisexual than men from other countries (42.8% vs 33.0%, p<0.001). Among CEE GUM patients, STI diagnoses (excluding HIV) were more likely among men than women, those under 25 years old, from an A2 country and who were recorded as homosexual or bisexual (table 1). In multivariate analysis, gender and age remained statistically significant. Seven CEE GUM patients (0.06% of all CEE GUM patients) were diagnosed with HIV during the study period, five of whom were men recorded as homosexual or bisexual.

SALLEE cross sectional survey

A total of 299 men and women attending these clinics completed the SALLEE questionnaire.

The SALLEE GUM sample therefore represents 24.4% (299/1225) of all CEE GUM patients who attended the clinics (excluding sessions for FSWs) over the data collection period. 325 CEE GUM patients were approached and 26 declined to take part, giving a response rate of 92%.

Comparing SALLEE GUM and SALLEE community samples

In order to explore the factors which may lie behind attendance at the GUM clinic, we examined differences between the SALLEE GUM and the SALLEE community samples (table 3). Women in the SALLEE community sample were more likely than men to have attended a GUM clinic in the UK (7.6% vs 4.5%, p=0.002). In comparison to community respondents, male and female GUM respondents were younger, more educated and had been in the UK for longer. They were less likely to be married or co-habiting. Female GUM respondents were more likely to be working than their community counterparts. Overall, SALLEE GUM respondents were substantially more likely than community respondents to report at least one new sexual partner in the past year (women: 67.9% vs 28.3%, p<0.001; men: 75.6% vs 45.1%, p<0.001). Although male community respondents were more likely to report one or more new heterosexual partners than their GUM counterparts (44.4% vs 31.5%, p=0.017), SALLEE GUM respondents were more likely than community respondents to report one or more homosexual partners in the past five years and the difference was particularly marked among men (54.3% vs 1.8%, p<0.001).

Community men were more likely to report consistent heterosexual condom use than GUM men (38.7% vs 13.5%, p=0.002) but there was no difference on consistency of condom use between women in the two samples (28.4% vs 29.9%, p=0.716). Community men were also more likely to have paid for sex with a woman (35.2% vs 24.3%, p=0.063). Among men reporting male sexual partners, however, there was no difference between the samples on ever paying for sex with a man (13.5% vs 12.0%, p=0.858). Three quarters of female and male community respondents reported assortative heterosexual sexual mixing (whereby their most recent partner was from their home country), compared to 25.9% of female GUM respondents and 56.5% of male GUM respondents (p<0.001 for female and male comparisons). GUM men were much less likely than community men to report a most recent male partner from their home country (9.6% vs 58.3%, p<0.001).

Male and female SALLEE GUM respondents were more likely than community respondents to report a previous STI diagnosis, being paid for sex in the UK and recreational drug use (all p<0.01). There were no differences between the samples on injecting drug use.

In logistic regression analysis adjusting for background characteristics (age, work status, education, relationship status, time in the UK, country of birth), all significant associations between sample and the sexual risk behaviours listed in table 3 remained significant (at p<0.001 among women and p<0.01 among men). The only exception was recreational drug use among men with an adjusted odds ratio of 1.55, p=0.063.

DISCUSSION

CEE patients make up a notable minority of patients in two central London GUM clinics.

Previous patient data from these GUM clinics (May 2006-April 2007) showed that 2.2% of men and 6.6% of women were from an A8 country. [START_REF] Burns | Increased attendances by people of Eastern European origin at sexual health services in London[END_REF] In this paper, we included men and women from the two CEE countries which joined the EU in January 2007, giving them entitlement to NHS services, and found that CEE men now make up 2.9% and CEE women make up 7.0% of patients.

The key factors associated with having a new STI diagnosis among CEE patients were gender and age. Surveillance data from the UK as a whole also show that people under 25 years old are disproportionately affected by STIs. [START_REF]Sexually Transmitted Infections and Young People in the United Kingdom: 2008 report[END_REF] A previous STI was reported by forty percent of SALLEE GUM respondents which is consistent with data from the broader population of people attending GUM clinics in Britain. One third of men and forty percent of women who had attended a GUM clinic in Britain reported a previous STI diagnosis, [START_REF] Fenton | Reported sexually transmitted disease clinic attendance and sexually transmitted infections in Britain: prevalence, risk factors, and proportionate population burden[END_REF] as did forty-one percent of those attending one of the GUM clinics included in the present study. [START_REF] Hughes | Comparison of risk factors for four sexually transmitted infections: results from a study of attenders at three genitourinary medicine clinics in England[END_REF] Half of the women attending GUM sessions for FSWs were from CEE countries and almost half of these were Romanian. While sessions specifically for sex workers were excluded from our analysis, sex workers attending general GUM clinics may have contributed towards increased new STI diagnoses among people from A2 countries. As a newer migrant population, people from A2 countries may be more likely to use GUM services only if they have a suspected STI whereas A8 patients might be more likely to come for a sexual health check. We are unable to test these hypotheses as data on sex work and time in the UK are not routinely collected.

CEE MSM appear to be more likely to access GUM services than their heterosexual counterparts.

Male CEE patients were more likely to be recorded as homosexual or bisexual than men from other countries. Homosexuality is no longer a criminal offence in CEE countries but stigmatization and discrimination are widely reported in the region, [START_REF]HIV and other STIs among MSM in the European Region[END_REF][START_REF][END_REF] which may encourage higher levels of migration among CEE men who have sex with men (MSM) compared to the CEE population as a whole.

Data collected from SALLEE respondents suggest that GUM patients have increased risk due to higher turnover of sexual partners, more homosexual partnerships among men and commercial sex. This is consistent with data showing that increased partner numbers and same sex partnerships are more commonly reported among people who have attended GUM clinics across Britain. [START_REF] Fenton | Reported sexually transmitted disease clinic attendance and sexually transmitted infections in Britain: prevalence, risk factors, and proportionate population burden[END_REF] Levels of consistent condom use were similar for women across both samples but the higher turnover of sexual partners among GUM respondents is likely to put them at greater risk for acquisition and transmission of STIs. Recreational drug use, which was highest among male GUM respondents, was found to be substantially higher among MSM compared to heterosexual men and women at a London HIV testing clinic. [START_REF] Bolding | Heterosexual men and women who seek sex through the Internet[END_REF] Interestingly, male SALLEE GUM respondents were less likely to have paid for sex with a woman than men in the community whereas paying for sex is associated with GUM clinic attendance in Britain. [START_REF] Fenton | Reported sexually transmitted disease clinic attendance and sexually transmitted infections in Britain: prevalence, risk factors, and proportionate population burden[END_REF] GUM services may not be reaching all CEE men who are at risk for STIs.

A major point of concern is that all self-reported HIV and the majority of previous STIs among SALLEE respondents had been diagnosed in the UK. We cannot rule out the possibility that infections went undiagnosed in their countries of origin or the impact of an age effect unrelated to migration on sexual risk behaviours among this group of young migrants. However, other factors are likely to have played a part. Firstly, migration has been identified as an independent risk factor for HIV. [START_REF] Brockerhoff | Migration, sexual behaviour and the risk of HIV in Kenya[END_REF] These CEE migrants have taken a risk in coming to the UK and it is thought that such risk-taking may filter through to other areas of their lives. [START_REF]Migrants' Right to Health[END_REF] Secondly, the sexual norms in the UK may be less limiting than in their countries of origin. Thirdly, data suggest that the incidence of STIs may be higher in the UK than in CEE countries: notification rates of gonorrhoea and Chlamydia are higher in the UK than in CEE countries and the UK is only exceeded in new HIV diagnoses by Estonia and Latvia. [START_REF]Annual Epidemiological Report on Communicable Diseases in Europe[END_REF] It also has the highest rate of newly diagnosed cases of HIV infection among MSM in Europe. [START_REF] Likatavičius | An increase in newly diagnosed HIV cases reported among men who have sex with men in Europe, 2000-6: implications for a European public health strategy[END_REF] SALLEE GUM respondents were much less likely to report assortative sexual mixing than men and women in the community and this may have increased their risk for acquisition of STIs.

A key limitation of our study is that we did not to link data from the cross sectional survey to clinic records which means that we are unable to look at the association between reported sexual behaviour and a new STI diagnosis. We wanted to assure respondents of anonymity and our high response rate suggests that we were successful in this respect although other studies have found that respondents are willing to consent to linking questionnaire data to their clinic records. [START_REF] Mercer | How much do delayed healthcare seeking, delayed care provision, and diversion from primary care contribute to the transmission of STIs?[END_REF] There were some data missing on nationality although this did not vary according to whether patients were or were not diagnosed with a new STI. CEE migrants come from ten different countries across an area of wide regional variation but the small size of our study sample did not provide sufficient statistical power to analyse behavioural data at the country level. The limitations of convenience samples have been published [START_REF] Evans | The sexual attitudes and lifestyles of London's Eastern Europeans (SALLEE Project): design and methods[END_REF] but it is reassuring that our SALLEE GUM sample did not differ from the CEE GUM population on age, gender or nationality. While HIV positive patients were excluded from our analysis of GUM patient data, seven SALLEE GUM respondents reported that they were HIV positive. However, inclusion of HIV positive SALLEE GUM respondents provides a more complete picture of CEE migrants accessing GUM services.

These findings have important implications for cross-border transmission of infections. Our data show that sexual mixing with people from the UK and other countries is one of the factors associated with GUM clinic attendance, a risk indicator for STIs. In addition to frequent trips home, CEE migrants often stay in the UK for short but recurring periods,[14] creating an opportunity for the transmission of infections between countries. Language barriers, confusion over entitlement and lack of knowledge about the services provided by GUM clinics may mean that migrants do not fully benefit from public health programmes or services. Bulgarians and Romanians may also benefit from specific targeting, given the increased likelihood of STI diagnosis among A2 GUM patients and the high proportion of Romanian women at GUM sessions for sex workers. CEE migrants are entitled to NHS services and the UK needs to ensure they are aware of sexual health services in the UK and able to access them.

Word count: 3051

  July 2008 -31 st March 2009). Recruitment in the clinics was mainly undertaken by two members of the research team, one of whom was a native speaker of Polish and Lithuanian. The SALLEE community sample was recruited from a range of venues in London over the same period. The nine fieldworkers involved in community sample recruitment were native speakers of six of the languages of the CEE countries.

Figure 1a :

 1a Figure 1a: Previous STI diagnoses reported by male SALLEE GUM respondents, by country of diagnosis

Table 1 : Factors associated with having a new STI diagnosis † among CEE patients attending two GUM clinics in London n % p value Crude OR (95% CI) p value Adjusted OR § (95% CI) p value

 1

	All	165	13.5	-	-	-	-	-
	Gender
	Female	92	10.9		1.00		1.00
	Male	73	19.2	<0.001	1.94 (1.39, 2.71)	<0.001	2.06 (1.38, 3.10)	<0.001
	Age
	25 years and over	100	11.7		1.00		1.00
	Under 25 years	65	17.7	0.005	1.62 (1.16, 2.28)	0.005	1.85 (1.30, 2.63)	0.001
	Country of birth
	A8	128	12.6		1.00		1.00
	A2	37	17.5	0.057	1.47 (0.99, 2.20)	0.058	1.37 (0.91, 2.06)	0.136
	Recorded as homosexual or bisexual
	Not homo / bisexual	133	12.6		1.00		1.00
	Homo / bisexual	32	18.8	0.028	1.61 (1.05, 2.46)	0.029	1.08 (0.65, 1.79)	0.780

† STI diagnoses include syphilis; gonorrhoea; Chlamydia; HSV (1st attack); genital warts (1st attack); trichomonas, uncomplicated non-gonococcal / non-specific urethritis; and complicated non-specific infection (non-chlamydial / non-gonococcal) including pelvic inflammatory dosease and epididymitis § adjusting for the other variables listed in this table

Table 2 : Background characteristics of SALLEE GUM respondents compared to all CEE GUM patients attending between 1 st June 2008 and 31 st March 2009

 2 Two thirds of SALLEE GUM respondents were female (68.2%). The majority had been in the UK for more than one year (this was more likely among people from the A8 than from the A2 (94.5% vs 74.5%, p<0.001)), and had returned home twice or more in the previous year (68.5%). SALLEE GUM respondents were similar in age to the population of CEE GUM patients who attended the clinics during the data collection period (27.0 vs 27.4 p=0.218) (table 2), were as likely to be male (31.8% vs 31.1%, p=0.823) and the distribution of CEE nationalities was similar (p=0.167), with Poles making up about forty percent of the total.A previous STI diagnosis was reported by 38.0% of male SALLEE GUM respondents and 41.2% of females (p=0.607). HIV positive status was reported by 2.2% of men (2/93) and 1.0% of women (2/195). All HIV positive respondents had been diagnosed in the UK and the majority of previous STI diagnoses had been made in the UK (fig 1aand 1b).

		SALLEE GUM respondents n = 299	All CEE GUM patients n = 1225	p-value
		n	%	n	%
	Age in years: mean (SD)	27.0 (4.7)		27.4 (5.2)		0.218
	Male	95	31.8	381	31.1	0.823
	Country of birth					0.167
	Bulgaria	24	8.1	82	6.7
	Czech Republic	15	5.1	107	8.7
	Estonia	3	1.0	26	2.1
	Hungary	20	6.7	103	8.4
	Latvia	18	6.1	54	4.4
	Lithuania	36	12.1	111	9.1
	Poland	126	42.4	487	39.8
	Romania	32	10.8	129	10.5
	Slovakia	22	7.4	118	9.6
	Slovenia	1	0.3	8	0.7

Table 3 : Background characteristics and sexual risk behaviour among SALLEE respondents, by gender and sample

 3

	Women

STIs, sex work and drug use

	Ever had STI (excl HIV & thrush)	80	41.2	118	11.4	<0.001	35	38.0	82	8.8	<0.001
	Been paid for sex in UK	15	7.7	6	0.5	<0.001	8	8.5	15	1.4	<0.000
	Recreational drug use (last year)	63	31.2	124	10.8	<0.001	41	43.6	311	28.7	0.003
	Ever injected drugs	5	2.5	26	2.2	0.841	4	4.3	57	5.3	0.672
	Injected drugs (last year)	2	1.0	12	1.0	0.949	3	3.2	22	2.0	0.455

AI = anal intercourse;

excluding men who did not have AI with their male sexual partners and including women who had sex with women;

among men and women who have had heterosexual sex in the past

weeks;[START_REF]Sexually Transmitted Infections and Young People in the United Kingdom: 2008 report[END_REF] among men who have had sex with a woman;

among men who have had sex with a man

Acknowledgements:

We are grateful for the guidance provided by our expert and community advisory boards, and for the hard work and commitment of the fieldworkers who collected the data. We would also like to thank the staff and management at the Mortimer Market Centre and the Archway Sexual Health Clinic; the many commercial, social and educational premises that facilitated recruitment for our fieldworkers; and everyone who took part in the study. This study was supported by the MRC Sexual Health and HIV Research Strategy Committee and the North Central London Research Consortium (NoCLoR) Grant Enhancement Fund. The views expressed are those of the authors and not necessarily those of the MRC, NoCLoR or the Health Departments. Contributors: FB, CM, CG, RM, JI, GH participated in the design of this study. AE, VP and FB were responsible for study coordination and data collection. AE conducted the statistical analysis and was the lead writer of this paper. All authors have contributed to the drafting of the manuscript and have seen and approved the final version.

article (if accepted) to be published in STI and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence: http://group.bmj.com/products/journals/instructions-for-authors/licence-forms.

Standard statistical tests such as chi-square and the Student's t-test were used to examine associations between factors. Logistic regression modelling was used to obtain odds ratios (OR) and adjusted odds ratios (aOR). Analysis was performed using SPSS12.0 (SPSS Inc, Chicago, Illinois, USA).

Ethics

The study was granted approval from the Camden and Islington Community Research Ethics Committee (07/H0722/110).

Conflicts of Interest: No conflicts of interest

Funding: This study was supported by the MRC Sexual Health and HIV Research Strategy Committee and the North Central London Research Consortium (NoCLoR) Grant Enhancement Fund. The sponsor of the study had no role in study design, the collection, analysis or interpretation of the data, the writing of the report or the decision to submit the paper for publication.

Copyright licence statement:

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this


M.D.; Manuel Pablos

M.D.; María-Antonia Remacha

J.-M Rodríguez-Calleja

Ph.D Jesús A Santos

Ph.D.; Andrés Otero

M.-L García-López

Dr María-Luisa García-López

Ph.D Jose M Rodríguez-Calleja

Ph.D María-Luisa García-López

Jose-María Rodríguez-Calleja

A Santos

María-Luisa García-López

Professor María-Luisa García-López

IDENTITY, VIRULENCE GENES AND CLONAL RELATEDNESS OF Aeromonas ISOLATES FROM PATIENTS WITH DIARRHEA AND DRINKING WATER

Keywords: Aeromonas, stool specimens, drinking water, PFGE Aeromonas, stool specimens, drinking water, PFGE 34

HAL is

Introduction 35

Aeromonas spp. are primarily aquatic organisms that may be readily isolated 36 from aquatic environments, drinking water and a wide range of foods. Some species 37 cause disease in fish, amphibians and reptiles. In humans, mesophilic Aeromonas are 38 recognized as causative agents of extraintestinal diseases such as septicemia, [START_REF] Seshadri | Genome sequence of Aeromonas 478 hydrophila ATCC 7966T: jack of all trades[END_REF] particularly in immunocompromised hosts, wound infections and a number of less well 40 described diseases [START_REF] Janda | Evolving concepts regarding the genus Aeromonas: 434 an expanding panorama of species, disease presentations, and unanswered 435 questions[END_REF]. Over the past 25 years, mesophilic Aeromonas have received 41 increasing attention as an emergent agent of food borne illness. In Spain, data from the 42 National Epidemiology Centre [START_REF]Epidemiological comment on 395 reported diseases and Microbiological Information System. Year 2006. Spain. 396 Boletín Epidemiológico[END_REF] indicate that Aeromonas spp. occupied the fourth 43 place among causes of total gastrointestinal diseases reported each year over the period 44 1997 to 2006. Because Aeromonas spp. can occur in municipal drinking water supplies 45 despite water treatment strategies, the possibility of waterborne Aeromonas infections in 46 humans has been discussed [START_REF] Joseph | Update on the genus Aeromonas[END_REF][START_REF]Guidelines for drinking-water quality. 539 Addendum: microbiological agents in drinking water[END_REF]. For this reason, some countries such as the [START_REF] Talon | Clonal identification of Aeromonas 509 hydrophila strains using randomly amplified polymorphic DNA analysis[END_REF] Netherlands have set standards for Aeromonas spp. in drinking water [START_REF] Anonymous | Besluit van 9 januari 2001 tot wijziging van het 352 waterleidingbesluit in verband met de richtlijn betreffende de kwaliteit van voor 353 menselijke consumptie bestemd water. 1-53[END_REF]. 48 Pathogenesis of Aeromonas spp. involves a wide group of virulence 49 determinants, their roles not being conclusively established for any human infections. [START_REF] Vilches | Complete type III secretion system of a 523 mesophilic Aeromonas hydrophila strain[END_REF] As for other bacteria, it appears that only certain intraspecific subsets of strains act as 51 human pathogens [START_REF] Joseph | Update on the genus Aeromonas[END_REF][START_REF] Von Graevenitz | The role of Aeromonas in diarrhea: a review[END_REF]. The literature shows that the occurrence of genes encoding 52 hemolytic, cytotoxic, cytotonic and enterotoxic activities (aerA, hlyA, alt, and ast) may 53 contribute to diarrheal-related virulence [START_REF] Heuzenroeder | Distribution of two 426 hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: 427 correlation with virulence in a suckling mouse model[END_REF][START_REF] Sha | Role of various enterotoxins in 485 Aeromonas hydrophila-induced gastroenteritis: generation of enterotoxin gene-486 deficient mutants and evaluation of their enterotoxic activity[END_REF][START_REF] Wong | Inactivation of two 535 haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a 536 suckling mouse model[END_REF] and that expression of peritrichous 54 flagella encoded by the laf gene cluster enhance eukaryotic cells adherence and 55 invasiveness, and also the ability to form biofilms in vitro [START_REF] Gavin | Lateral 410 flagella are required for increased cell adherence, invasion and biofilm 411 formation by Aeromonas spp[END_REF]. Moreover, the presence 56 of a type III secretion system (TTSS) can play a role in the virulence of motile 57

Aeromonas by delivering a range of proteins (including toxins) into the host cell [START_REF] Yu | A 543 type III secretion system is required for Aeromonas hydrophila AH-1 544 pathogenesis[END_REF]. In 58 similar to Shiga-like toxins of verotoxigenic E. coli has been observed [START_REF] Haque | 422 Diarrheal and environmental isolates of Aeromonas spp. produce a toxin similar 423 to Shiga-like toxin 1[END_REF][START_REF] Snowden | Prevalence of environmental Aeromonas in South East Queensland, 497 Australia: a study of their interactions with human monolayer Caco-2 cells[END_REF]. 60

This study was designed with two aims. The first was to determine the 61 prevalence and the identification of identity of Aeromonas species in cases of 62 gastroenteritis in a Spanish city (León, NW of the country) and to investigate the 63 presence of virulence-associated genes in isolates from patients with diarrhea. The 64 second aim was to establish the genetic relationships of strains from stool specimens 65 and from León drinking water by using pulsed-field gel electrophoresis (PFGE). 66 Germany). After incubation at 28 ºC for 22 h, a total of 160 suspect colonies were 73 screened for standard phenotypic traits: Gram stain, motility, oxidase and catalase 74 production, fermentation of glucose and resistance to vibriostatic agent O/129 [START_REF] Kirov | Aeromonas and Plesiomonas[END_REF]. 75 Drinking water isolates. For comparative typing by using pulse field gel 76 electrophoresis (PFGE), we included 12 A. caviae and A. media strains (Table 3) which 77 were isolated during the study period from León municipal drinking water meeting 78 quality standards for indicator organisms and chlorine levels [START_REF] Anonymous | Real Decreto 140/2003, de 7 de febrero, por el que se 356 establecen los criterios sanitarios de la calidad del agua de consumo humano. 357[END_REF]. 79

Materials And Methods 67

Phenotypic characterization of Aeromonas isolates from patients with 80

diarrhea. A total of 32 Aeromonas isolates (one from each positive sample) was tested 81 for indole production, gas from glucose, Voges Proskauer, L-arginine hydrolysis, lysine and ornithine decarboxylase, esculin hydrolysis, susceptibility to 30 g of cephalotin, 83 methyl red test, acid production from m-inositol, L-arabinose, lactose, D-mannitol, 84 salicin, D-sucrose, D-rhamnose, and D-sorbitol, gelatinase and elastase activities, and -85 hemolysis on sheep blood agar plates as described elsewhere [START_REF] González-Rodríguez | Foodborne pathogenic bacteria in prepackaged fresh retail portions of 415 farmed rainbow trout and salmon stored at 3 ºC[END_REF]. The phenotypic 86 scheme of Abbott et al. [START_REF] Abbott | The genus Aeromonas: 339 biochemical characteristics, atypical reactions, and phenotypic identification 340 schemes[END_REF] was employed for identification of isolates to Aeromonas 87 Groups level. Additional tests useful in differentiating species within the A. hydrophila 88 complex (A. hydrophila, A. bestiarum and A. salmonicida), the A. caviae complex (A. 89 caviae, A. media and A. eucrenophila), and the Group 1 "ODC (ornithine 90 decarboxylase) + only" (A. veronii biovar veronii and A. allosaccharophila) were also 91 performed [START_REF] Abbott | The genus Aeromonas: 339 biochemical characteristics, atypical reactions, and phenotypic identification 340 schemes[END_REF]. The suicide phenomenon at 30 and 37 ºC was investigated as described 92 by Namdari and Cabelli [START_REF] Namdari | The suicide phenomenon in motile aeromonads[END_REF]. 93 Detection of associated virulence genes. The target genes investigated in this 94 study, the sequence of each primer, the annealing temperatures, the expected PCR 95 products and their references are listed in Table 1. 96 Genomic DNA was extracted from exponential cultures of the organisms in 97

Trypticase soy broth (Becton Dickinson S.A., Madrid, Spain) taking into account ISO 98 22174 general requirements and according to ISO 20837 [START_REF] Anonymous | Microbiology of food and animal feeding 359 stuffs. Polymerase chain reaction (PCR) for the detection of food-borne 360 pathogens. General requirements and definitions[END_REF][START_REF]Microbiology of food and animal feeding 363 stuffs. Polymerase chain reaction (PCR) for the detection of food-borne 364 pathogens. Requirements for sample preparation for qualitative detection[END_REF]. PCR amplification and 99 detection of PCR products were performed according to the general requirements 100 described in ISO 22174 and ISO 20838 [START_REF] Anonymous | Microbiology of food and animal feeding 359 stuffs. Polymerase chain reaction (PCR) for the detection of food-borne 360 pathogens. General requirements and definitions[END_REF][START_REF] Anonymous | Microbiology of food and animal feeding 367 stuffs. Polymerase chain reaction (PCR) for the detection of food-borne 368 pathogens. Requirements for amplification and detection for qualitative 369 methods[END_REF]. PCR conditions for the aerA, hlyA, alt, 101 ast, laf, ascF-G, stx1 and stx2 genes have been described elsewhere [START_REF] Chacón | Type III 382 secretion system genes in clinical Aeromonas isolates[END_REF][START_REF] Martínez | Foodborne and indicator bacteria in farmed molluscan shellfish before 449 and after depuration[END_REF][START_REF] Paton | Detection and characterization of Shiga toxigenic 458 Escherichia coli by using multiplex PCR assays for stx 1 , stx 2[END_REF][START_REF] Rodríguez-Calleja | Rabbit meat as a source of bacterial foodborne pathogens[END_REF] between fingerprints was calculated with the Dice coefficient. Cluster analysis was 117 performed using the unweighted pair-group method with average linkages (UPGMA). 118

Isolates were considered genetically "indistinguishable" if their PFGE patterns were 119 100% similar and "closely related" if they showed more than 85% similarity, 120 corresponding to one to three band differences [48]. Isolates with PFGE profiles 121 showing less than 85% similarity were defined as being genetically unrelated strains. A 122 similarity threshold of >85% defined clusters. 123

Results 124

Of the 800 stool samples evaluated, 32 samples from different patients were 125 positive for Aeromonas spp. with an overall positivity rate of 4%. There were 17 126 positive samples when the mean monthly environmental temperature was below 12 ºC 127 and 15 when the mean monthly environmental temperature ranged between 12 and 24.3 128 ºC (http://clima.meteored.com/clima-en-Europa.html). 129

Information about patients and Aeromonas isolates recovered from their stools is 130 shown in Table 2. All positive samples except one were from outpatients. Of the 32 131 selected Aeromonas isolates (one from each patient), 26 were allocated to the A. caviae 132 complex, four to the A. hydrophila complex and two to the "ODC+ only" Group 1. 133

The species found were A. caviae [START_REF] Haque | 422 Diarrheal and environmental isolates of Aeromonas spp. produce a toxin similar 423 to Shiga-like toxin 1[END_REF] patients had acute diarrhea without vomiting, two had chronic diarrhea, and three had 143 diarrhea, vomits and fever. Stool samples from pediatric patients (up to 14 years old) 144 and males accounted for 68.7% and 56.2% of the Aeromonas isolates, respectively. The 145 27 patients with acute diarrhea were infected with strains of the five Aeromonas species 146 found, 12 of them being simultaneously positive for other enteric pathogens (Table 2). 147

Patients with chronic diarrhea and those with diarrhea, vomits and fever were infected 148 with A. caviae alone and one with A. hydrophila alone. 149

The distribution of associated virulence genes among the Aeromonas diarrheal 150 isolates is summarized in Table 3. None of the strains harbored the genes ascF-G, stx1 151 or stx2 and nine bore plasmids. -hemolysis on sheep blood agar plates was only 152 displayed by isolates carrying the aerA and/or the hlyA genes, the remaining isolates 153 showing zones of incomplete hemolysis. 154 Two human isolates (H8 and H9) were consistently non-typeable with the PFGE 155 applied method neither by using a second enzyme (SpeI). PFGE following XbaI 156 digestion identified 30 patterns whose profiles differed by at least one band. The 157 dendrogram obtained (Fig. 1) showed a cluster (cluster B) of eight A. media water 158 strains with 100% similarity. The remaining water isolates (A. caviae) grouped together 159 in cluster G, two of them (W73 and W74) being indistinguishable from strain H13 160 recovered from a 51 years old female, without apparent underlying medical 161 complications, who was negative for other enteric pathogens. There were two clusters 162 with two human isolates with identical XbaI PFGE profiles: two identified as A. 163 bestiarum (cluster A) and two as A. caviae (cluster F). Other clusters (C, D, E and H) 164 included nine closely related A. caviae isolates from patients with diarrhea. 165 Indistinguishable isolates possessed identical virulence genes. 166 Discussion 167

One of the most commonly reported human infections by Aeromonas spp. is 168 gastroenteritis. Clinical manifestations of disease can range from mild self-limiting 169 watery diarrhea to a more severe and invasive dysenteric form. Chronic diarrheal 170 episodes and isolated cases of a cholera-like illness have also been described [START_REF] Janda | Evolving concepts regarding the genus Aeromonas: 434 an expanding panorama of species, disease presentations, and unanswered 435 questions[END_REF]. In 171 several reported studies throughout the world, Aeromonas spp. have been isolated at a 172 rate of 0.6 to 7.2% from patients with diarrhea predominantly from infants and children 173 [START_REF] Albert | Prevalence of enterotoxin 349 genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, 350 and the environment[END_REF][START_REF] Borchardt | Aeromonas isolates from 374 human diarrheic stool and groundwater compared by pulsed-field gel 375 electrophoresis[END_REF][START_REF] Figura | Prevalence, 405 species differentiation, and toxigenicity of Aeromonas strains in cases of 406 childhood gastroenteritis and in controls[END_REF][START_REF] Moyer | Clinical significance of Aeromonas species isolated from 451 patients with diarrhea[END_REF][START_REF] Sinha | Prevalence, serotype distribution, antibiotic susceptibility and 492 genetic profiles of mesophilic Aeromonas species isolated from hospitalized 493 diarrhoeal cases in Kolkata, India[END_REF][START_REF] Wilcox | Aeromonas spp. as a 533 potential cause of diarrhoea in children[END_REF]. The highest rate, 52.4%, was detected in Peruvian infants with 174 severe diarrhea although 57.6% of the Aeromonas-positive patients had mixed 175 infections with other enteric pathogens [START_REF] Pazzaglia | High frequency of coinfecting enteropathogens in 463 Aeromonas-associated diarrhea of hospitalized Peruvian infants[END_REF]. A similar high frequency of co infection 176 has been found in Bangladesh and India [START_REF] Albert | Prevalence of enterotoxin 349 genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, 350 and the environment[END_REF][START_REF] Sinha | Prevalence, serotype distribution, antibiotic susceptibility and 492 genetic profiles of mesophilic Aeromonas species isolated from hospitalized 493 diarrhoeal cases in Kolkata, India[END_REF]. Our results are in agreement with those 177 found in most of the above surveys. Thus, Aeromonas spp. was isolated from 4.0% of 178 the patients, most of them being infants (46.9%) and other pediatric patients (21.9%). 179 Clinically, acute illness was the most common symptom whereas fever, vomiting or 180 chronic diarrhea were present in only a few cases. In addition, common enteric 181 pathogens other than Aeromonas spp. were recovered from more than one third of 182 diarrheic stools. Because of the ubiquitous nature of Aeromonas spp. in raw foods and 183 water, it has been argued that in mixed infections these bacteria are transient colonizer 184 without causal relationship with disease, but also that multiple pathogens may act 185 synergistically to produce diarrhea [START_REF] Albert | Prevalence of enterotoxin 349 genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, 350 and the environment[END_REF][START_REF] Pazzaglia | High frequency of coinfecting enteropathogens in 463 Aeromonas-associated diarrhea of hospitalized Peruvian infants[END_REF]. Unlike most surveys that have found peaks 186 of Aeromonas-associated gastroenteritis in warmer months, we did not observe seasonal 187 patterns for isolation. 188

The majority of our isolates (71.9%), which were identified to phenospecies with 189 biochemical schemes [START_REF] Abbott | The genus Aeromonas: 339 biochemical characteristics, atypical reactions, and phenotypic identification 340 schemes[END_REF] and later confirmed by molecular methods (AFLP and gyrB 190 gene sequences; data not shown), belonged to the species A. caviae. There also were 191 some isolates of A. media, A. hydrophila, A. bestiarum and A. veronii biovar veronii. In 192 several studies conducted in Europe, the United States and India, A. caviae was 193 dominant followed by A. hydrophila and A. veronii biovar sobria [START_REF] Albert | Prevalence of enterotoxin 349 genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, 350 and the environment[END_REF][START_REF] Borchardt | Aeromonas isolates from 374 human diarrheic stool and groundwater compared by pulsed-field gel 375 electrophoresis[END_REF][START_REF] Figura | Prevalence, 405 species differentiation, and toxigenicity of Aeromonas strains in cases of 406 childhood gastroenteritis and in controls[END_REF][START_REF] Moyer | Clinical significance of Aeromonas species isolated from 451 patients with diarrhea[END_REF][START_REF] Sinha | Prevalence, serotype distribution, antibiotic susceptibility and 492 genetic profiles of mesophilic Aeromonas species isolated from hospitalized 493 diarrhoeal cases in Kolkata, India[END_REF][START_REF] Wilcox | Aeromonas spp. as a 533 potential cause of diarrhoea in children[END_REF]. 194

This agrees with an earlier report from Spain [START_REF] Velasco-Muñoz | Aeromonas 516 infection surveillance in Spain. Years 1989-1999. Spain. Boletín 517 Epidemiológico[END_REF], which noted that, between 1989 and 195 1999, the three species accounted for 90% of all Aeromonas-associated gastroenteritis. 196

Other studies in Brazil, Thailand and India [START_REF] Kannan | Isolation and identification of aeromonas from 441 patients with acute diarrhoea in Kolkata, India[END_REF][START_REF] Von Graevenitz | The role of Aeromonas in diarrhea: a review[END_REF] reported A. hydrophila as 197 predominant, and a high isolation rate of A. trota has also been found [START_REF] Albert | Prevalence of enterotoxin 349 genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, 350 and the environment[END_REF]. These 198 differences may depend on the geographical location but also on other factors at least in 199 part related to the isolation and identification methods used. Thus, as most Aeromonas 200 culture media contain ampicillin, the high incidence of A. trota, an ampicillin-201 susceptible species, found by the latter authors was attributed to the isolation technique 202 used. 203 A. media is isolated from the environment and occasionally from patients with 204 diarrhea [START_REF] Albert | Prevalence of enterotoxin 349 genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, 350 and the environment[END_REF], but its clinical significance is not known [START_REF] Kirov | Aeromonas and Plesiomonas[END_REF] although Singh [START_REF] Singh | A putative heat-labile enterotoxin expressed by strains of 489 Aeromonas media[END_REF] reported 205 that regardless of their source, A. media strains are potentially enterotoxigenic. In this 206 study, two of the A. media positive patients had concurrent infection with Salmonella. 207 A. veronii biovar veronii and A. bestiarum have rarely been isolated from humans and 208 little information is available regarding its occurrence in clinical specimens; however, 209 two strains of each species were isolated by us as the sole pathogen from two children 210 and two elderly men, and also from gastroenteritis in Switzerland, India and Brazil [14, 211 25, 43]. 212

The pathogenicity of Aeromonas spp. is multifactorial and complex and may 213 involve the products of a number of different genes acting either alone or in concert [19, 214 40]. One rational approach to determine whether Aeromonas strains have the potential 215 to be virulent is to screening for an array of virulence-related genes [START_REF] Aguilera-Arreola | Virulence potential and genetic 344 diversity of Aeromonas caviae, Aeromonas veronii, and Aeromonas hydrophila 345 clinical isolates from Mexico and Spain: a comparative study[END_REF][START_REF] Wang | Detection and characterization of the 529 hemolysin genes in Aeromonas hydrophila and Aeromonas sobria by multiplex 530 PCR[END_REF]. A potent 216 virulence factor of Aeromonas strains is the cytotoxic enterotoxin Act, which is closely 217 related to aerolysin (AerA), both of them being hemolytic, cytotoxic, enterotoxic and 218 lethal for mice. Aeromonas may also produce heat labile (Alt) and heat stable (Ast) 219 cytotonic enterotoxins [START_REF] Galindo | Host immune 408 responses to Aeromonas virulence factors[END_REF]. Sha et al. [START_REF] Sha | Role of various enterotoxins in 485 Aeromonas hydrophila-induced gastroenteritis: generation of enterotoxin gene-486 deficient mutants and evaluation of their enterotoxic activity[END_REF] established a role for the three enterotoxins 220 in causing gastroenteritis in a mouse model by developing isogenic mutants with the 221 major contribution from Act, followed by the Alt and Ast. Results from Albert et al. [START_REF] Albert | Prevalence of enterotoxin 349 genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, 350 and the environment[END_REF] 222 suggest that the product of both the alt and ast genes may act synergistically to induce 223 severe diarrhea. 224

The incidence of the aerA, alt and ast genes in our clinical isolates was 25, 71.9 225 and 18.8%, respectively, the alt + /ast + combination being detected in four of them 226 (12.5%). These findings are comparable with those of Sinha et al. [START_REF] Sinha | Prevalence, serotype distribution, antibiotic susceptibility and 492 genetic profiles of mesophilic Aeromonas species isolated from hospitalized 493 diarrhoeal cases in Kolkata, India[END_REF] who, in isolates 227 from Indian patients with acute diarrhea, found that act/aerA accounted for 30%, alt 228 was dominant and ast was the less prevalent. Overall, among fecal isolates from Mexico tested by us, that enable them to overcome host defenses and establish infection. Among 256 these factors are surface molecules, different toxins, many enzymes, iron acquisition 257 systems, protein secretion systems and novel virulence determinants that may play an 258 important role in pathogenesis [START_REF] Galindo | Host immune 408 responses to Aeromonas virulence factors[END_REF][START_REF] Seshadri | Genome sequence of Aeromonas 478 hydrophila ATCC 7966T: jack of all trades[END_REF][START_REF] Sha | 481 Surface-expressed enolase contributes to the pathogenesis of clinical isolate SSU 482 of Aeromonas hydrophila[END_REF]. 259

Our results and those from others suggest that in Aeromonas clinical isolates the 260 distribution pattern of associated-virulence genes varies depending on the species and 261 geographical location and also that despite current knowledge of Aeromonas virulence 262 determinants, there is still no way to identify a given clinical or environmental isolate 263 that has the capacity to cause human illness although some data indicate a role of both 264 the bacterial virulence factors and host-responses [START_REF] Galindo | Host immune 408 responses to Aeromonas virulence factors[END_REF]. 265

The presence of TTSS seems to be related to the pathogenicity of some 266

Aeromonas strains [START_REF] Vilches | Complete type III secretion system of a 523 mesophilic Aeromonas hydrophila strain[END_REF][START_REF] Yu | A 543 type III secretion system is required for Aeromonas hydrophila AH-1 544 pathogenesis[END_REF]. However, it is more frequently found in A. hydrophila and 267

A. veronii strains than in A. caviae isolates, even from clinical specimens [START_REF] Vilches | Complete type III secretion system of a 523 mesophilic Aeromonas hydrophila strain[END_REF]. This fact 268 can explain the lack of detection of TTSS genes in our isolates, as they belong mainly to 269 the species A. caviae. 270 Human infections with Aeromonas spp. have occasionally been associated with 271 hemolytic uremic syndrome (HUS) [START_REF] Figueras | 401 Aeromonas hemolytic uremic syndrome. A case and a review of the literature[END_REF], a life-threatening condition normally 272 associated with enterohemorragic E. coli (EHEC) strains producing the cytotoxins Stx1 273 and/or Stx2. The stx1 gene has been detected in clinical and environmental isolates of A. 274 hydrophila, A. caviae and A. veronii biovar sobria [START_REF] Haque | 422 Diarrheal and environmental isolates of Aeromonas spp. produce a toxin similar 423 to Shiga-like toxin 1[END_REF][START_REF] Snowden | Prevalence of environmental Aeromonas in South East Queensland, 497 Australia: a study of their interactions with human monolayer Caco-2 cells[END_REF]. As this could explain 275 reports on HUS related to Aeromonas spp., we investigated the presence of stx1 and 276 stx2 genes and plasmids. None of our isolates were PCR positive for the stx genes and 277 28% harbored plasmids. Although most strains of A. salmonicida carry at least one large 278 plasmid, plasmids are detected less commonly (15 to 30%) in human diarrhea-279 associated Aeromonas isolates [START_REF] Brown | Plasmids and Aeromonas virulence[END_REF]. 280 13 PFGE, which has remarkable discriminatory power and reproducibility, has been 281 successfully applied to the subtyping of many pathogenic bacteria [START_REF] Barrett | Interpretation of pulsed-field 371 gel electrophoresis patterns in foodborne disease investigations and surveillance. 372[END_REF]. Over the last 282 years, a number of studies using PFGE and other molecular methods have been used for 283 comparative typing of clinical and/or environmental Aeromonas strains [10, 15, 32, 45-284 47] most of them concluding: (i) that specific clones colonized certain aquatic 285 ecosystems such as drinking water supplies probably as consequence of biofilms 286 development, (ii) that there was not similarity among clinical isolates, and (iii) that 287 stools and drinking water isolates were genetically unrelated. In one of the earliest 288 studies, following a number of cases of diarrhea in children using a small community 289 water supply, Moyer et al. [START_REF] Moyer | Application 453 of ribotyping for differentiating aeromonads isolated from clinical and 454 environmental sources[END_REF] reported colonization of the water supply with 290 Aeromonas identical ribotypes, but unique ribotypes in Aeromonas patient strains, 291 concluding that water was not the source of Aeromonas infections. Similar results were 292 obtained in French studies using RAPD and PFGE for typing isolates from hospital-293 acquired infections and from the hospital water supply [START_REF] Talon | Pulsed-505 field gel electrophoresis as an epidemiological tool for clonal identification of 506 Aeromonas hydrophila[END_REF][START_REF] Talon | Clonal identification of Aeromonas 509 hydrophila strains using randomly amplified polymorphic DNA analysis[END_REF]. De la Morena et al. [START_REF] De La Morena | 392 Diarrhea associated with Aeromonas species in children in day care centers[END_REF] 294 studied Aeromonas isolates recovered from children involved in two outbreaks of 295 gastroenteritis in day-care centers, finding that the PFGE patterns differed in all except 296 two strains. Also by PFGE, Aeromonas isolates from patients with diarrhea and 297 groundwater isolates from private wells throughout Wisconsin were genetically 298 unrelated [START_REF] Borchardt | Aeromonas isolates from 374 human diarrheic stool and groundwater compared by pulsed-field gel 375 electrophoresis[END_REF]. By using RAPD, REP-PCR and ERIC-PCR, Szczuka and Kaznowski 299 [START_REF] Szczuka | Typing of clinical and environmental 500 Aeromonas sp. strains by random amplified polymorphic DNA PCR, repetitive 501 extragenic palindromic PCR, and enterobacterial repetitive intergenic consensus 502 sequence PCR[END_REF] found that strains isolated from patients in Poland, different parts of Europe and 300 Asia were different clones and that there was not genetic similarity between clinical and 301 environmental strains although the persistence of a single clone in a city water 302 distribution was observed. 303

In this study, PFGE analysis of the water isolates revealed persistence of two 304 indistinguishable or closely related (up to three band differences) clones of A. media and caviae water isolates with identical pattern of associated-virulence genes (aerA -/hlyA - 315 /alt + /ast -/laf + ) showed identical pulsotype providing a link between Aeromonas spp. and 316 diarrheal disease. Chopra [START_REF] Chopra | Characterization of waterborne Aeromonas species for their 385 virulence potential[END_REF] also reported that two pairs of A. caviae/A.media isolates 317 from water and patients carrying only one of the three enterotoxin genes (alt) had 318 indistinguishable PFGE patterns, the author indicating that biochemical identification of 319 isolates, determination of virulence traits and DNA fingerprinting could provide 320 necessary information on the pathogenic potential of Aeromonas spp. that can cause 321 waterborne infections. 322

In summary, infection with Aeromonas spp., mainly A. caviae, occurred 323 throughout the year and was significantly more common in pediatric patients. Acute 324 diarrhea was the predominant clinical feature, with the majority of isolates harboring 325 associated-virulence genes. PFGE of human and water isolates, which were recovered 326 from the same geographic area during the same time period, revealed genetic 327 relatedness and persistence over time in water and some clinical isolates. Interestingly, 328 one human isolate had indistinguishable PFGE pattern and identical virulence pattern 329 than two water isolates, suggesting waterborne infection. 330 Letters together strain number represent: W, isolates from water samples and H, isolates from clinical stools

  of plasmid encoded expression by Aeromonas isolates of cytotoxins 59

Fecal specimens .

 specimens From October 2005 to January 2007, 800 stool specimens 68 submitted by Primary Care Centers to the Hospital of León Microbiology and 69 Parasitology Service (coverage population, 351.086 inhabitants) were routinely tested 70 for the detection of enteric pathogens (bacteria, enteric viruses and parasites) and also 71 plated on freshly prepared m-Aeromonas selective agar (Merck, KGaA, Darmstadt, 72

Formatted

Formatted:Fig. 1

 1 Fig. 1 Genetic similarity based on PFGE of Aeromonas strains from patients with diarrhea and drinking water. The horizontal upper bar represents genetic similarity (%).

  The standardized PulseNet PFGE protocol for pathogenic Gram-negative 109 enteric bacteria[36] and a CHEF-DR III apparatus (Bio-Rad) were used for PFGE 110 analysis. DNA plugs were digested with XbaI restriction enzyme (New England 111 Biolabs, Ipswich, MA, USA). Strains were typed at least at two separate occasions. 112 After electrophoresis, the gels were stained with ethidium bromide, and documented 113 with the Kodak Electrophoresis Documentation and Analysis System 290 (EDAS 290, 114 Kodak Co., Rochester, NY). The banding patterns were analyzed with the GelCompar II 115 program (version 5.1., Applied Maths, St.-Martens-Latem, Belgium). Similarity 116

	107	manufacturer's instructions and analyzed by agarose gel electrophoresis (1.5%) at 100 V
	108	for 40 min.
		PFGE.
	102	.
	103	Aliquots from each reaction were analyzed by 2% agarose gel electrophoresis and
	104	viewed after ethidium bromide staining under UV light.
		Plasmids detection. Plasmid DNA isolation was performed with the Quantum 6

14

  A. caviae in the water distribution system of León over the[START_REF]Epidemiological comment on 395 reported diseases and Microbiological Information System. Year 2006. Spain. 396 Boletín Epidemiológico[END_REF] months study period. For 306 clinical isolates, different PFGE patterns were present in stool specimens, some of them 307 grouping in clusters of indistinguishable or closely related strains. Interestingly, there 308 was no clear relation among the origin of isolates or time of isolation; for instance, the 309 two A. caviae isolates in cluster C were recovered in October 2005 and May 2006, those 310 in cluster D in February and October 2006 and those in cluster E in April 2006 and 311 January 2007. The indistinguishable isolates grouped in clusters A and F were detected 312 in the same months (March and June 2006) but from samples obtained in four different 313 Primary Care Centers. It is also noteworthy that one A. caviae clinical isolate and two A. 314

Table 1

 1 PCR primers, annealing temperatures and expected PCR products for 547

	548	virulence associated genes
	Target	Primers´ sequences (5´-3´)	Annealing	Produced	References
	Genes		temperature	amplicon
			(ºC)	length (bp)
	aerA	GC(A/T)GA(A/G)CCC(A/G)TCTATCC(A/T)G	55	252	Santos et al. [38]
		TTTCTCCGGTAACAGGATTG
	hlyA	GGCCGGTGGCCCGAAGATACGGG	62	597	Wong et al. [54]
		GGCGGCGCCGGACGAGACGGG
	alt	CCA TCC CCA GCC TTT ACG CCA T	63	338	Martínez et al. [30]
		TTT CAC CGA GGT GAC GCC GT
	ast	ATG CAC GCA CGT ACC GCC AT	66	260	Martínez et al. [30]
		ATC CGG TCG TCG CTC TTG GT
	laf	GGT CTG CGC ATC CAA CTC	60	550	Gavín et al. [20]
		GCT CCA GAC GGT TGA TG
	ascF-G ATG AGG TCA TCT GCT CGC GC	55	ca 1 Kb Chacón, 2004 [12]
		GGA GCA CAA CCA TGG CTG AT

Field Code Changed

Prep Plasmid Miniprep kit (Bio-Rad Laboratories, Hercules, CA) according to the

and Catalonia (Spain), Aguilera-Arreola et al.[START_REF] Aguilera-Arreola | Virulence potential and genetic 344 diversity of Aeromonas caviae, Aeromonas veronii, and Aeromonas hydrophila 345 clinical isolates from Mexico and Spain: a comparative study[END_REF] reported that genes for the aerolysin-

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Acknowledgments 331

This work was supported by Spanish CICYT (grant AGL2004-04672-ALI) and 332 Junta de Castilla y León (Ayuda Grupo de Excelencia GR155). Manuel Pablos was 333 beneficiary of a fellowship from MEC. 334

We thank Borja Linage for help in PFGE testing. We also appreciate the kind 335 collaboration of Dr F. Cachón (Microbiology and Parasitology Service, Hospital of 336 León). 337