Céline Langendorf
email: celine.langendorf@epicentre.msf.org

Simon Le Hello

Aissatou Moumouni

Malika Gouali

Abdoul- Aziz Mamaty

Rebecca F Grais

François-Xavier Weill
email: francois-xavier.weill@pasteur.fr

Anne-Laure Page

Enteric Bacterial Pathogens in Children with Diarrhea in Niger: Diversity and Antimicrobial Resistance

Background

Although rotavirus is the leading cause of severe diarrhea among children in sub-Saharan Africa, better knowledge of circulating enteric pathogenic bacteria and their antimicrobial resistance is crucial for prevention and treatment strategies.

Methodology/Principal Findings

As a part of rotavirus gastroenteritis surveillance in Maradi, Niger, we performed stool culture on a sub-population of children under 5 with moderate-to-severe diarrhea between April 2010 and March 2012. Campylobacter, Shigella and Salmonella were sought with conventional culture and biochemical methods. Shigella and Salmonella were serotyped by slide agglutination. Enteropathogenic Escherichia coli (EPEC) were screened by slide agglutination with EPEC O-typing antisera and confirmed by detection of virulence genes. Antimicrobial susceptibility was determined by disk diffusion. We enrolled 4020 children, including 230 with bloody diarrhea. At least one pathogenic bacterium was found in 28.0% of children with watery diarrhea and 42.2% with bloody diarrhea. Mixed infections were found in 10.3% of children. EPEC, Salmonella and Campylobacter spp. were similarly frequent in children with watery diarrhea (11.1%, 9.2% and 11.4% respectively) and Shigella spp. were the most frequent among children with bloody diarrhea (22.1%). The most frequent Shigella serogroup was S. flexneri (69/122, 56.5%). The most frequent Salmonella serotypes were Typhimurimum (71/355, 20.0%), Enteritidis (56/355, 15.8%) and Corvallis (46/ 355, 13.0%). The majority of putative EPEC isolates was confirmed to be EPEC (90/111, 81.1%). More than half of all Enterobacteriaceae were resistant to amoxicillin and co-trimoxazole. Around 13% (46/360) Salmonella exhibited an extended-spectrum beta-lactamase phenotype.

Introduction

Although diarrhea mortality rate among children under 5 years of age in sub-Saharan Africa has been decreasing annually by about 4% since 2000, mortality remains high [START_REF] Liu | Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000[END_REF]. Of the estimated 3.6 million child deaths in 2010, 12% were attributed to diarrheal diseases. The incidence of childhood diarrhea in Africa has also decreased from 4.2 to 3.3 episodes per child-year from 1990 to 2010, but sub-Saharan Africa still accounts for one third of diarrheal episodes yearly (500 million of 1.7 billion worldwide), with the highest incidence among children 6-11 months of age [START_REF] Walker | Diarrhea incidence in low-and middle-income countries in 1990 and 2010: a systematic review[END_REF].

While rotavirus is the leading cause of severe diarrhea among children under 5 [START_REF] Sanchez-Padilla | Burden of disease and circulating serotypes of rotavirus infection in sub-Saharan Africa: systematic review and meta-analysis[END_REF][START_REF] Waggie | Review of rotavirus studies in Africa: 1976-2006[END_REF][START_REF] Mwenda | Burden and epidemiology of rotavirus diarrhea in selected African countries: preliminary results from the African Rotavirus Surveillance Network[END_REF], with about 30% of diarrhea cases being rotavirus positive in sub-Saharan Africa [START_REF] Walker | Global burden of childhood pneumonia and diarrhoea[END_REF], many other viruses, parasites and bacteria cause serious diarrheal morbidity and mortality but are not vaccine preventable [START_REF] Kotloff | Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study[END_REF][START_REF] Sire | Community-acquired infectious diarrhoea in children under 5 years of age in Dakar, Senegal[END_REF][START_REF] Bonkoungou | Bacterial and viral etiology of childhood diarrhea in Ouagadougou, Burkina Faso[END_REF][START_REF] Phan | Acute diarrhea in West African children: diverse enteric viruses and a novel parvovirus genus[END_REF][START_REF] Nitiema | Burden of rotavirus and other enteropathogens among children with diarrhea in Burkina Faso[END_REF][START_REF] Moyo | Age specific aetiological agents of diarrhoea in hospitalized children aged less than five years in Dar es Salaam, Tanzania[END_REF]. Among bacterial etiologies, typical enteropathogenic Escherichia coli (EPEC) are associated with a higher risk of mortality in infants 0-11 months of age with moderate-to-severe diarrhea and Shigella is the first or second cause of moderate-to-severe diarrhea among children 1 to 5 years old [START_REF] Kotloff | Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study[END_REF][START_REF] Lanata | Global causes of diarrheal disease mortality in children <5 years of age: a systematic review[END_REF].

The Sahel (an ecoregion lying between the Sudanian savanna to the south and the Sahara desert to the north) bears the burden of some of the highest rates of diarrhea, malnutrition and malaria among children under 5 [START_REF] Liu | Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000[END_REF]. The vicious cycle involving diarrheal disease and malnutrition places this population at higher risk of morbidity and mortality [START_REF] Kotloff | Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study[END_REF][START_REF] Guerrant | Malnutrition as an enteric infectious disease with long-term effects on child development[END_REF][START_REF] Opintan | Pediatric diarrhea in southern Ghana: etiology and association with intestinal inflammation and malnutrition[END_REF]. Moreover, some diarrhea-causing agents like non-typhoidal Salmonella (NTS) were found to be the dominant contributor to invasive bacterial disease with high mortality in Africa [START_REF] Tabu | Differing burden and epidemiology of non-Typhi Salmonella bacteremia in rural and urban Kenya, 2006-2009[END_REF][START_REF] Feasey N A, Dougan | Invasive non-typhoidal Salmonella disease: an emerging and neglected tropical disease in Africa[END_REF][START_REF] Page | Infections in children admitted with complicated severe acute malnutrition in Niger[END_REF]. Several studies in the Sahelian region showed that multi-drug resistant organisms have been emerging over the last decades, with the general spread of resistance to amoxicillin, co-trimoxazole and chloramphenicol [START_REF] Laxminarayan | Antibiotic resistancethe need for global solutions[END_REF][START_REF] Okeke | Antimicrobial resistance in developing countries. Part I: recent trends and current status[END_REF][START_REF] Vlieghe | Antibiotic resistance among bacterial pathogens in Central Africa: a review of the published literature between 1955 and 2008[END_REF][START_REF] Ahmed | Antimicrobial agent resistance in bacterial isolates from patients with diarrhea and urinary tract infection in the Sudan[END_REF][START_REF] Bercion | Distribution and antibiotic susceptibility of Shigella isolates in Bangui, Central African Republic[END_REF][START_REF] Brooks | Surveillance for bacterial diarrhea and antimicrobial resistance in rural western Kenya, 1997-2003[END_REF][START_REF] Djie-Maletz | High rate of resistance to locally used antibiotics among enteric bacteria from children in Northern Ghana[END_REF]. Resistance to fluoroquinolones and extended-spectrum cephalosporins (ESCs) in Enterobacteriaceae have also been reported to a lower extent [START_REF] Bercion | Increasing prevalence of antimicrobial resistance among Enterobacteriaceae uropathogens in Bangui, Central African Republic[END_REF][START_REF] Raji | Point-surveillance of antibiotic resistance in Enterobacteriaceae isolates from patients in a Lagos Teaching Hospital, Nigeria[END_REF][START_REF] Harrois | Prevalence and characterization of extended-spectrum β-lactamase-producing clinical Salmonella enterica isolates in Dakar, Senegal, from 1999 to 2009[END_REF][START_REF] Tansarli | Proportion of extended-spectrum β-lactamase (ESBL)-producing isolates among Enterobacteriaceae in Africa: evaluation of the evidence-systematic review[END_REF]. Data on the prevalence and antimicrobial resistance of the main circulating enteropathogenic bacteria remain limited in the Sahel and in Niger in particular, due to the lack of laboratory facilities. Yet, better knowledge of circulating endemic and epidemic enteric pathogens and their antimicrobial resistance is crucial for prevention, including vaccines against Shigella and diarrheagenic E. coli [START_REF] Das | Vaccines for the prevention of diarrhea due to cholera, Shigella, ETEC and rotavirus[END_REF][START_REF] Kim | Shigella vaccine development: prospective animal models and current status[END_REF] and treatment strategies with antibiotics.

Here, we describe the main enteric and possibly pathogenic bacteria isolated from the stools of children under 5 with moderate-to-severe diarrhea included in rotavirus gastroenteritis surveillance in Maradi, Niger between April 2010 and March 2012.

Materials and Methods

Study sites

Surveillance was set up as described previously [START_REF] Page | Rotavirus surveillance in urban and rural areas of niger, april 2010-march 2012[END_REF] in the region of Maradi, located approximately 500 km away from the capital city Niamey. One regional hospital and 10 health centers were included in four different health districts: Maradi (1 regional hospital), Madarounfa (3 health centers), Aguié (3 health centers) and Guidan Roumdji (4 health centers). After an initial period of one year of surveillance, surveillance was interrupted in the health district of Aguié. The analysis presented here was restricted to a 24-month period from April 2010 to March 2012.

Study population

Children were included in the general rotavirus surveillance study if they were aged 0 to 59 months, consulting at a study site with watery diarrhea and signs of moderate or severe dehydration, and if the parent or legal guardian accepted participation in the study. Children with reported bloody diarrhea were included during the second year [START_REF] Page | Rotavirus surveillance in urban and rural areas of niger, april 2010-march 2012[END_REF]. A sub-sample of the children included in the general rotavirus surveillance was selected to participate in the sub-study on bacterial etiology of diarrhea. These children were: the first child included daily with watery diarrhea at each health center; the first 2 children included daily with watery diarrhea at the regional hospital; all included children with reported bloody diarrhea and producing stool specimens.

Data and specimen collection

A standardized questionnaire was used to collect socio-demographic characteristics (sex, age), clinical presentation (dehydration, fever, vomiting), medical care (rehydration treatment, hospitalization, inclusion in a nutritional program) and outcomes.

At the health centers, stool specimens were collected using sterile plastic containers and systematically tested for rotavirus using Vikia Rota-Adeno rapid test (bioMérieux, Marcy l'Etoile, France) [START_REF] Page | Rotavirus surveillance in urban and rural areas of niger, april 2010-march 2012[END_REF]. Stool samples were then transferred onto Cary-Blair medium (Oxoid, Basingstoke, UK) using sterile swab and stored at +2-8°C until transported in cold containers to the Epicentre laboratory in Maradi city every other day. At the Maradi regional hospital, we maintained the fresh stools at +2-8°C before transfer to the laboratory within 1 hour after sampling.

Stool culture, bacterial identification and antimicrobial susceptibility testing

The bacterial pathogens sought included Salmonella spp, Shigella spp, EPEC, and, starting from July 2010, Campylobacter spp. Upon arrival at the laboratory, swabs from the Cary-Blair tubes or fresh stools were plated onto desoxycholate citrate lactose media (DCL, Bio-Rad, Marnes-la-Coquette, France), Hektoen media (HK, Bio-Rad), Butzler media (Oxoid) and Muller-Kauffmann broth (MK, AES Chemunex, Combourg, France). DCL, HK and MK were incubated at 37°C in normal atmosphere. Butzler media were incubated for 48h in microaerophilic atmosphere using atmospheric generator (bioMérieux). After 18h incubation, 10μL of MK broth were inoculated onto Chromagar Salmonella media (ChromAgar, Paris, France) and incubated for an additional 18-24 h. After incubation, suspected colonies of Salmonella and Shigella spp. were submitted to metabolic identification using API strips (bioMérieux) and partially serogrouped by slide agglutination using polyvalent Salmonella O-typing antisera (Bio-Rad). From DCL media, between 1 and 10 lactose-fermenting colonies were picked and tested by slide agglutination with EPEC O-typing antisera (Bio-Rad). Twelve EPEC serogroups were targeted by the four polyvalent antisera used: I (O111, O55, O26), II (O86, O119, O127), III (O125, O126, O128), and IV (O114, O124, O142). These serogroups corresponded to those recognized as being EPEC by the World Health Organization (WHO) in 1987, with the exception of O124 which was replaced by O158 in the WHO definition [START_REF]Programme for control of diarrheal diseases (CDD/83.3 Rev1)[END_REF]. Reactive colonies were subsequently isolated and confirmed as E. coli using API strips. Campylobacter isolates were identified by colony aspect on Butzler media, Gram stain examination, oxidase (+) and catalase (+). The sodium hippurate hydrolysis test was then performed to differentiate C. jejuni from C. coli.

Antimicrobial susceptibility of each isolate was determined by the disk diffusion method on Mueller-Hinton agar (+5% sheep blood for Campylobacter spp) as recommended by the Antibiogram Committee of the French Society for Microbiology (CA-SFM) [34]. The panel of the 13 antimicrobials tested (Bio-Rad) was amoxicillin (AMX, 25μg), amoxicillin-clavulanic acid (AMC, 20/10μg), cefalotin (CF, 30μg), cefotaxime (CTX, 30μg), ceftazidime (CAZ, 30μg), cefepime (FEP, 30μg), imipenem (IPM, 10μg), nalidixic acid (NA, 30μg), ciprofloxacin (CIP, 5μg), co-trimoxazole (trimethoprim-sulfamethoxazole, SXT, 1,25/23,75μg), gentamicin (GM, 15μg), amikacin (AN, 30μg), and erythromycin (for Campylobacter spp. only, E, 15UI). Extendedspectrum β-lactamase (ESBL) phenotype was assessed by using the double-disk synergy (DDS) method [34] on isolates displaying resistance or intermediate resistance to ESCs. The isolates displaying resistance to ciprofloxacin by disk diffusion were confirmed by determination of the minimal inhibitory concentration (MIC) using Etest strips (bioMérieux). CA-SFM breakpoints were used for disk diffusion and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used for MIC. E. coli strain CIP 76.24 (ATCC 25922) was used as a quality control strain.

Typing of Shigella and Salmonella

Salmonella (n = 370) and Shigella (n = 138) isolates were stored at Epicentre laboratory then sent to the French National Reference Center for E. coli, Shigella and Salmonella (FNRC-ESS), Institut Pasteur, Paris. Species identification was confirmed using conventional methods and serotyping was done by slide agglutination assays using a complete set of antisera allowing recognition of all described Salmonella [START_REF] Grimont | Antigenic formulae of the Salmonella serotypes[END_REF] and Shigella [START_REF] Bopp | Escherichia, Shigella, and Salmonella[END_REF] serotypes. Typing antisera were purchased from Bio-Rad, Eurobio (Les Ulis, France), Statens Serum Institute (Copenhagen, Denmark), Sifin (Berlin, Germany) or were homemade at the FNRC-ESS. Two serotype Kentucky isolates were typed by multilocus sequence typing (MLST) as described previously [START_REF] Hello | International spread of an epidemic population of Salmonella enterica serotype Kentucky ST198 resistant to ciprofloxacin[END_REF].

Characterization of EPEC isolates

All putative EPEC isolated from rotavirus-positive children (n = 37) and a random sample of putative EPEC isolated from rotavirus-negative children in 2010 (n = 74) were sent to the FNRC-ESS for confirmation and serotyping. All isolates were subjected to the following biochemical tests: motility, glucose (acid and gas), H 2 S, indole production, lysine decarboxylase, ornithine decarboxylase, glycerol, lactose, d-mannitol, and sorbitol. DNA was extracted from all phenotypically confirmed E. coli isolates with the InstaGene matrix kit (Bio-Rad). PCR was used to detect the uidA gene encoding the beta-glucuronidase; thus confirming the assignment of the isolates to E. coli and to detect several virulence genes associated with EPEC and other diarrheagenic E. coli. The targeted virulence genes were (i) eae (for EPEC attaching and effacing), which encodes intimin, an outer membrane adhesin essential for the intimate attachment of the EPEC or enterohemorrhagic E. coli (EHEC) to enterocytes, (ii) bfpA (for bundle-forming pilus) carried by the EPEC adherence factor (EAF) plasmid found in typical EPEC, (iii) stx1 and stx2 encoding Shiga toxins, markers of EHEC, (iv) EHEC-hlyA encoding the hemolysin of EHEC, (v) in case of eae negativity, aggR, a transcriptional activator of aggregative adherence fimbriae expression in enteroaggregative Escherichia coli (EAEC), (vi) and in case of eae and aggR negativity, lt and st genes of enterotoxigenic E. coli (ETEC) and ial and ipah genes of enteroinvasive E. coli (EIEC). The different primers and basic amplification conditions are described in S1 Table [START_REF] Bej | Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid[END_REF][START_REF] Beaudry | Genotypic and phenotypic characterization of Escherichia coli isolates from dogs manifesting attaching and effacing lesions[END_REF][START_REF] Gunzburg | Identification of enteropathogenic Escherichia coli by PCRbased detection of the bundle-forming pilus gene[END_REF][START_REF] Lin | Detection of various variant verotoxin genes in Escherichia coli by polymerase chain reaction[END_REF][START_REF] Pollard | Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction[END_REF][START_REF] Paton | Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157[END_REF][START_REF] Boisen | New adhesin of enteroaggregative Escherichia coli related to the Afa/Dr/AAF family[END_REF][START_REF] Moseley | Isolation and nucleotide sequence determination of a gene encoding a heat-stable enterotoxin of Escherichia coli[END_REF][START_REF] Woodward | Detection of entero-and verocyto-toxin genes in Escherichia coli from diarrhoeal disease in animals using the polymerase chain reaction[END_REF][START_REF] Frankel | Multi-gene amplification: simultaneous detection of three virulence genes in diarrhoeal stool[END_REF]. All E. coli isolates that contained at least one virulence gene were serogrouped by slide agglutination using at first polyvalent I (O26, O44, O114, O125, O142, O158), II (O55, O86, O91, O111, O119, O126, O127, O128), III (O25, O78, O103, O118, O124, O145, O157, O164) and A (O1, O2, O18, O78) antisera from Sifin; then if needed (i.e., in case of an agglutination with a polyvalent antiserum) the corresponding monovalent antisera, also from Sifin. Not serotypable E. coli isolates (rough phenotype due to the complete or partial loss of lipopolysaccharide and leading to autoagglutination or no agglutination with the panel of antisera) were O-typed by a molecular method based on the analysis of the O-antigen gene cluster (rfb restriction fragment length polymorphism [rfb-RFLP]) after DNA extraction with the InstaGene matrix kit (Bio-Rad) [START_REF] Coimbra | Identification of Escherichia coli O-serogroups by restriction of the amplified O-antigen gene cluster (rfb-RFLP)[END_REF].

Data analysis

Double data entry was done using EpiData 3.1 (EpiData, Odense, Denmark), and data analysis using Stata 12.1 (College Station, Texas, USA). Severe diarrhea was defined using the 20-point Vesikari scale with a score 11 [START_REF] Lewis | Vesikari Clinical Severity Scoring System Manual[END_REF]. We performed a weighted analysis by month and district of inclusion to take into account the selection process when estimating the proportion and seasonality of the bacterial pathogens. For Salmonella and Shigella spp., only the isolates that were confirmed at FNRC-ESS are considered in this analysis.

Ethical Considerations

The study protocol was approved by the National Ethical Committee of Niger's Ministry of Public Health (Ref n°02/2009/CCNE) and by the Comité de Protection des Personnes, Ile-de-France XI, France. Participation was voluntary. An informed consent statement was read aloud in the local dialect before consenting parent or legal guardian signed or fingerprinted (procedures approved by both ethical committees). All children received treatment free of charge.

Results

Characteristics of the study population

In total, 8993 children were included in the rotavirus surveillance study from April 2010 to March 2012. Of these, we excluded 117 because of a delay between admission and testing of more than 3 days, 4 without clinical signs of dehydration, and 37 who sought care 14 days after onset of diarrhea. The 8835 remaining comprised 8566 children with watery diarrhea, of which 3790 (44.3%) were selected for the bacteriological study; and 269 with bloody diarrhea, of which 230 (85.5%) were also included in the bacteriological study.

Due to the selection process of children for the bacteriological study (i.e. inclusion of the first child per day in all health centers and first 2 children per day at Maradi hospital), children included in Maradi district (i.e. at the Maradi regional hospital), hospitalized children and intravenous treatment are over-represented compared to the initially enrolled population (Table 1). Other characteristics, such as sex, age, presence of fever, inclusion in a nutritional program and mortality were similar to the initial population (Table 1). Children with bloody diarrhea showed significantly less vomiting and a lower proportion of severe diarrhea according to the Vesikari score, compared to those with watery diarrhea (Table 1).

Proportion of pathogenic bacteria, seasonal distribution and clinical characteristics

Overall, at least one pathogenic bacterium was found in 1060 (28.0%) children with watery diarrhea and 97 (42.2%) children with bloody diarrhea. EPEC, Salmonella spp. and Campylobacter spp. were similarly frequent in children with watery and bloody diarrhea and Shigella spp. were the most frequent among children with bloody diarrhea (Table 2). Mixed infections (i.e. association with rotavirus or any other bacteria) were found in 10.3% of children overall. Fiftyfour children (1.3%) with sterile stool culture after 48h were excluded from all the subsequent analyses.

When the weighted proportions were extrapolated to the number of diarrhea cases, the seasonal distribution of pathogens showed a consistent peak of EPEC in March to June, when 54% of EPEC diarrhea occurred annually. There was a peak of Salmonella spp. in October to January during the first year with 50% of all Salmonella diarrhea (mean 27 cases per month). This same amplitude was not found the second year (mean 13 cases per month) (S1 Fig. ).

While the proportion of EPEC and rotavirus decreased with age, the proportion of Salmonella spp. increased (Fig. 1). All demographic and clinical characteristics showed significant differences among infection categories with the exception of sex (Table 3). The main associations after exclusion of mixed-infections were the following: Campylobacter infections with moderate dehydration (p = 0.007); EPEC infections with vomiting (p = 0.011) and severe dehydration (p<0.001); Salmonella infections with severe dehydration (p<0.001), fever (p = 0.016) and inclusion in a nutrition program (p<0.001); Shigella infections with deaths (p = 0.003); finally rotavirus infections with vomiting (p<0.001) and with non-inclusion in a nutrition program (p<0.001), as described elsewhere [START_REF] Page | Rotavirus surveillance in urban and rural areas of niger, april 2010-march 2012[END_REF]. While all isolated Campylobacter were C. jejuni, diversity of the other pathogens is described below.

Shigella and Salmonella diversity

Of 143 isolates initially identified as Shigella, 138 were sent to the FNRC-ESS. The missing isolates were due to storage difficulties after strain identification. Ten (10) strains were not confirmed as Shigella spp., leaving 128 confirmed Shigella isolates. Of these, 6 were not serotypable (four were rough and two were non-agglutinable with the panel of typing antisera). Of the 122 serotyped isolates, the most frequent serogroups were S. flexneri (n = 69, 56.5%), followed by S. dysenteriae (n = 24, 19.7%), S. boydii (n = 21, 17.2%), and finally S. sonnei (n = 8, 6.6%). Two isolates belonged to S. dysenteriae type 1, the causative agent of epidemic dysentery. Details of serotypes identified are provided in Table 4.

Of the 383 isolates initially identified as Salmonella, 370 were sent to the FNRC-ESS. The missing isolates were due to storage difficulties after strain identification. Nine (9) strains were not confirmed as Salmonella spp., one could not be subcultured, leading to 360 confirmed Salmonella isolates. In addition, 7 were not serotypable (rough). Two cultures contained more than one serotype; a mixture of serotypes Serenli and Suelldorf in one and a mixture of serotypes Mbao and Herston in the other. Among the 355 Salmonella serotypes, the most frequently encountered were Typhimurimum (n = 71, 20.0%), Enteritidis (n = 56, 15.8%), and Corvallis (n = 46, 13.0%) (Table 5). Another 64 serotypes were identified with less than 20 isolates for each, including S. enterica serotype Typhi (two isolates), the agent of typhoid fever and two potential new serotypes belonging to subspecies enterica. These potential new serotypes with antigenic formulae 44:z:z6 (one isolate) and 47:d:1,5 (two isolates) were sent to the World Health Organization Collaborative Center for Reference and Research on Salmonella, Institut Pasteur, Paris for confirmation and incorporation into the White-Kauffmann-Le Minor scheme. One with antigenic formula 47:d:1,5 has be confirmed and named serotype Maradi, while the other is still under study.

Antimicrobial resistance

Around 2% (6/328) of Campylobacter jejuni were resistant to amoxicillin-clavulanic acid and erythromycin, with 5 isolates resistant to both. More than half of all Enterobacteriaceae, and up to 90% of EPEC isolates were resistant to amoxicillin and co-trimoxazole, while they were mostly sensitive to all other antibiotics tested (Table 6). Among Shigella isolates, S. flexneri (71.8%) were more resistant to amoxicillin than S. sonnei (12.5%), but similarly resistant to cotrimoxazole (86.0% in S. flexneri and 100% in S. sonnei). The two S. dysenteriae type 1 isolates were resistant to amoxicillin and susceptible to other tested drugs. Forty-six Salmonella and

Discussion

Almost one-third of children were infected with or carried a bacterial pathogen and around one in ten presented co-infections with either another bacterial pathogens or rotavirus, consistent with what has been found in other African countries [START_REF] Bonkoungou | Bacterial and viral etiology of childhood diarrhea in Ouagadougou, Burkina Faso[END_REF][START_REF] Moyo | Age specific aetiological agents of diarrhoea in hospitalized children aged less than five years in Dar es Salaam, Tanzania[END_REF]. Although it is widely recognized that isolation of a bacterial pathogen from stool does not directly imply that the diarrhea is attributable to this particular pathogen, the characterization of circulating agents in a population still provides important information for the prevention and control of these infections and can complement case-control studies on diarrhea etiology. In case-control studies, rotavirus and Shigella spp. are consistently reported as highly associated with diarrhea patients (cases), EPEC shows significant but intermediate association, while Salmonella spp. and Campylobacter are often found in similar proportions in patients with or without diarrhea [START_REF] Kotloff | Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study[END_REF][START_REF] Meng | Etiology of diarrhea in young children and patterns of antibiotic resistance in Cambodia[END_REF][START_REF] Swierczewski | Surveillance for enteric pathogens in a case-control study of acute diarrhea in Western Kenya[END_REF]. Here, it is thus difficult to conclude on the fractions of diarrhea cases attributable to each pathogen, except for Shigella spp., which is probably responsible for around 2% of non-bloody diarrhea and over 20% of bloody diarrhea in this area.

In our study, Shigella spp. were associated with the highest mortality overall and particularly affected children included in a nutritional program, with a case fatality ratio of 15%. Most Shigella spp. isolates were resistant to amoxicillin, although S. sonnei was mostly sensitive, similar to what was described in Senegal and Central African Republic [START_REF] Bercion | Distribution and antibiotic susceptibility of Shigella isolates in Bangui, Central African Republic[END_REF][START_REF] Sire | Antimicrobial resistance in Shigella species isolated in Dakar, Senegal (2004-2006)[END_REF]. Resistance to ESCs and fluoroquinolones was not found here, which is also consistent with the low prevalence in other reports from Africa and in contrast with the emergence of resistance in Asia [START_REF] Bonkoungou | Bacterial and viral etiology of childhood diarrhea in Ouagadougou, Burkina Faso[END_REF][START_REF] Sire | Antimicrobial resistance in Shigella species isolated in Dakar, Senegal (2004-2006)[END_REF][START_REF] Njuguna | Use of populationbased surveillance to define the high incidence of shigellosis in an urban slum in Nairobi, Kenya[END_REF][START_REF] Zhang | Wide dissemination of multidrug-resistant Shigella isolates in China[END_REF][START_REF] Holt | Tracking the establishment of local endemic populations of an emergent enteric pathogen[END_REF]. The isolated Shigella serotypes showed the typical pattern described in developing countries with a clear predominance of S. flexneri, in particular over S. sonnei [START_REF] Kotloff | Global burden of Shigella infections: implications for vaccine development and implementation of control strategies[END_REF]. Several Asian countries have recently experienced a transition from predominance of S. flexneri to S. sonnei, probably due to improvement of overall nutritional status, sanitation and socioeconomic status [START_REF] Ud-Din | Changing Trends in the Prevalence of Shigella Species: Emergence of Multi-Drug Resistant Shigella sonnei Biotype g in Bangladesh[END_REF][START_REF] Vinh | A changing picture of shigellosis in southern Vietnam: shifting species dominance, antimicrobial susceptibility and clinical presentation[END_REF][START_REF] Bangtrakulnonth | Shigella from humans in Thailand during 1993 to 2006: spatial-time trends in species and serotype distribution[END_REF]. The few studies available from sub-Saharan Africa suggest that it is not yet the case in this area [START_REF] Bonkoungou | Bacterial and viral etiology of childhood diarrhea in Ouagadougou, Burkina Faso[END_REF][START_REF] Sire | Antimicrobial resistance in Shigella species isolated in Dakar, Senegal (2004-2006)[END_REF][START_REF] Njuguna | Use of populationbased surveillance to define the high incidence of shigellosis in an urban slum in Nairobi, Kenya[END_REF][START_REF] Tiruneh | Serodiversity and antimicrobial resistance pattern of Shigella isolates at Gondar University teaching hospital, Northwest Ethiopia[END_REF], which emphasizes the need for vaccines including both species.

The isolation of S. dysenteriae type 1 from two children living in two different areas and 2 months apart indicates that this pathogen was still circulating in this area in 2011 but represented only 1.6% of all Shigella spp. and was not associated with outbreaks. The low level circulation of S. dysenteriae type 1 was also observed in a survey conducted in an urban slum in Nairobi, Kenya between 2007 and 2010 where S. dysenteriae type 1 represented 2.3% (6/262) of Shigella spp. isolated [START_REF] Njuguna | Use of populationbased surveillance to define the high incidence of shigellosis in an urban slum in Nairobi, Kenya[END_REF]. Although no outbreak due to S. dysenteriae type 1 has been reported in Africa since 2003-2004 [61], these studies document the low level circulation of Shiga's bacillus in two different regions of Africa where circumstances facilitating its transmission such as overcrowding, poor or lack of sanitation or health facilities might trigger local outbreaks.

Although screening for putative EPEC was based on O antigens (serogrouping) rather than on the presence of virulence genes, the eae gene, marker of EPEC was found in the vast majority of isolates, confirming the good correlation between serogroups and virulence. However, the identification method itself may be a limiting factor because it did not allow for the identification of other E. coli enteric pathovars, and in particular ETEC, which was identified as the cause of one third of the diarrhea attributable fraction (and first bacterial agent) in the GEMS study [START_REF] Kotloff | Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study[END_REF]. EAEC were also found as the most frequent bacterial pathogens in children with diarrhea in Africa, particularly in infants [START_REF] Bonkoungou | Bacterial and viral etiology of childhood diarrhea in Ouagadougou, Burkina Faso[END_REF][START_REF] Moyo | Age specific aetiological agents of diarrhoea in hospitalized children aged less than five years in Dar es Salaam, Tanzania[END_REF][START_REF] Karambu | Characterization and factors associated with diarrhoeal diseases caused by enteric bacterial pathogens among children aged five years and below attending Igembe District Hospital, Kenya[END_REF]. In our study, EPEC was associated with younger age, as well as vomiting and severe dehydration. It was not associated with a higher risk of dying, even in children less than 1 year (p = 0.11), in contrast to the 2.6 hazard ratio found in GEMS [START_REF] Kotloff | Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study[END_REF]. EPEC isolates presented the highest resistance to amoxicillin and co-trimoxazole, but were mostly sensitive to other antibiotics tested. The detection of one ESBL-producing EPEC warrants attention in the context of increasing resistance in Africa [START_REF] Tansarli | Proportion of extended-spectrum β-lactamase (ESBL)-producing isolates among Enterobacteriaceae in Africa: evaluation of the evidence-systematic review[END_REF][START_REF] Woerther | Massive Increase, Spread, and Exchange of Extended Spectrum {beta}-Lactamase-Encoding Genes Among Intestinal Enterobacteriaceae in Hospitalized Children With Severe Acute Malnutrition in Niger[END_REF].

We found that almost all EPEC (97.8%) were typical in agreement with other published data [START_REF] Trabulsi | Typical and atypical enteropathogenic Escherichia coli[END_REF]. Typical EPEC are associated with a human reservoir in low-income countries whereas atypical EPEC are closer to Shiga toxin-producing E. coli and can be found either in humans or animals of high-income countries. Interestingly, we identified typical EPEC belonging to a new serogroup or to a serogroup (O26) previously associated only with atypical EPEC [START_REF] Trabulsi | Typical and atypical enteropathogenic Escherichia coli[END_REF]. This could be due to a large diversity of EPEC populations circulating in low-income countries and not well-known due to the lack of local studies requiring molecular biology for EPEC confirmation.

Salmonella spp. were more frequently isolated in children included in a nutrition program, the proxy used to define the population of malnourished children in our study. This suggests that malnutrition could be a risk factor for Salmonella infection in general, and not only for invasive non-typhoidal Salmonella infection [START_REF] Feasey N A, Dougan | Invasive non-typhoidal Salmonella disease: an emerging and neglected tropical disease in Africa[END_REF]. The Salmonella peak around November 2010 corresponds to end of the annual malnutrition peak in Niger, and the number of children included in a nutritional program during this period in our study was decreasing (data not shown), suggesting that the 2 phenomena are temporally disconnected. This period also generally corresponds to the malaria peak in Niger, but we could not study possible associations between malaria and Salmonella isolation in diarrhea patients here since diagnosis of malaria was not recorded. In addition, although there was a slight increase in the number of Salmonella detected around November 2011, there was no clear peak and the seasonality of infection remains to be confirmed.

Despite a wide variety of Salmonella serotypes identified, around half of the isolates belonged to serotypes Typhimurium, Enteritidis and Corvallis. While the 2 first are known to be involved in a majority of diarrhea, as well as invasive NTS infections in Africa [START_REF] Feasey N A, Dougan | Invasive non-typhoidal Salmonella disease: an emerging and neglected tropical disease in Africa[END_REF][START_REF] Morpeth | Invasive non-Typhi Salmonella disease in Africa[END_REF][START_REF] Okoro | Intracontinental spread of human invasive Salmonella Typhimurium pathovariants in sub-Saharan Africa[END_REF][START_REF] Kariuki | Characterisation of community acquired non-typhoidal Salmonella from bacteraemia and diarrhoeal infections in children admitted to hospital in Nairobi, Kenya[END_REF], serotype Corvallis has not been described as a major serotype so far. The fact that almost all serotype Corvallis isolates showed very similar antimicrobial resistance profiles, including ESBL production, suggests that there was a clonal strain spreading in the area. This spread was disseminated, in both space and time since it was found in all districts and during the whole study period, with a peak in November 2010, along with other Salmonella spp. The comprehensive characterization of these isolates as well as others from older and more recent surveys is ongoing in order to elucidate the reservoir of this bacterial population. Two isolates of serotype Kentucky highly resistant to ciprofloxacin were isolated. They belonged to the multi-drug resistant S. enterica serotype Kentucky ST198-X1 strain which originated in Egypt during the 1990s, before disseminating across Africa, the Middle East, the Indian subcontinent and South East Asia beginning in the mid-2000s [START_REF] Hello | International spread of an epidemic population of Salmonella enterica serotype Kentucky ST198 resistant to ciprofloxacin[END_REF][START_REF] Hello | Highly drug-resistant Salmonella enterica serotype Kentucky ST198-X1: a microbiological study[END_REF].

Our study had several limitations. As mentioned above, the study was not designed to assess attributable fractions and it is difficult to conclude on the precise etiology of diarrhea in this context. Since the required case-control studies for such a purpose are time-and resource-consuming, knowledge of pathogen and site-specific odds ratio from multicentric studies such as GEMS would contribute to the interpretation of descriptive studies. In addition, the spectrum of bacterial pathogens sought did not include some important bacteria such as ETEC, EAEC, due to the lack of appropriate techniques (ie. PCR) to detect them on site. EHEC O157:H7, which can be detected by culture using appropriate media, was also not investigated here since it was not isolated in a previous study in the same area [START_REF] Page | Infections in children admitted with complicated severe acute malnutrition in Niger[END_REF]. Media for the detection of V. cholerae were available in case of suspicion of cholera, but were not used systematically.

In conclusion, this study provides updated information on enteric bacteria diversity and antimicrobial resistance in the Sahel region, where such data are scarce. Although rotavirus represents the first cause of diarrhea with dehydration, potentially pathogenic bacteria were found in a similar proportion of children (~30%), sometimes in co-infection. Whether they are or not the causative agent of diarrhea, bacterial infections and their antimicrobial resistance profiles should be considered in areas with a large proportion of malnutrition, which pre-disposes to severe and invasive infections. The emergence of ESBL-producing Salmonella spp. resistant to all locally available antimicrobials should particularly be monitored. The wide serotype diversity, in particular for Shigella spp., should also be considered for vaccine development.

EPEC

  .1371/journal.pone.0120275.t006 one EPEC isolate were resistant to ESCs and exhibited an ESBL phenotype. All the 46 ESBLproducing Salmonella belonged to serotype Corvallis. The majority of these isolates were also resistant to gentamicin (n = 31) and intermediate or resistant to ciprofloxacin (n = 23, MICs range 1-3 mg/L). Although there was a peak of ESBL-producing S. enterica serotype Corvallis in November 2010 (n = 13), the others were isolated throughout the study period. The district of origin of children with ESBL-producing Salmonella infection was more likely to be Maradi (41.3%, n = 19) compared to children with any Salmonella infection (12.0%, p<0.001). However, ESBL-producing S. enterica serotype Corvallis were also isolated from Guidan-Roumdji (n = 13), Madarounfa (n = 9) and other districts. Regarding resistance to ciprofloxacin, in addition to Corvallis isolates, only two other isolates were resistant (MICs 16 mg/L). Both isolates belonged to serotype Kentucky and were ST198 by MLST.

Table 1 .

 1 Socio-demographic and clinical characteristics of children with watery or bloody diarrhea, included in the bacteriology sub-study.

		Watery diarrhea	p*	Bloody diarrhea	p ‡
		(N = 3790)			(N = 230)
		n	%		n	%
	District of inclusion			<0.001			<0.001
	Maradi	658	17.4		37	16.1
	Madarounfa	1201	31.7		54	23.5
	Guidan Roumdji	1482	39.1		139	60.4
	Aguié	449	11.8		0	0.0
	Socio-demographic
	Male	2103	55.5	0.34	125	54.4	0.75
	Age, median [IQR]	9	[7-12]		12	[9-19]
	Clinical characteristics
	Severe dehydration	581	15.3	0.11	44	19.1	0.12
	Fever	852	22.5	0.53	62	27.0	0.12
	Vomiting	2461	65.0	0.006	105	46.1	<0.001
	Vesikari score 11	2759	72.9	0.54	128	55.7	<0.001
	Hospitalisation and outcome
	IV treatment	447	11.8	<0.001	21	9.1	0.22
	Hospitalisation	804	21.2	<0.001	39	17.0	0.13
	Included in a nutritional program	722	19.0	0.56	52	22.6	0.18
	Deaths	56	1.5	0.41	7	3.0	0.06

* Comparison with children with watery diarrhea included in the surveillance study but not included in the bacteriology study ‡ Comparison between children with watery or bloody diarrhea doi:10.1371/journal.pone.0120275.t001

Table 2 .

 2 Weighted proportion of enteric bacterial pathogens and rotavirus among children with watery and bloody diarrhea.

			Watery diarrhea			Bloody diarrhea
		n	%	95% CI	n	%	95% CI
	All
	Putative EPEC*	416	11.1	[10.1-12.2]	20	9.4	[6.1-13.2]
	Salmonella spp.	340	9.2	[8.2-10.3]	20	8.7	[5.8-13.4]
	Shigella spp.	78	2.0	[1.6-2.5]	50	22.1	[17.1-28.1]
	Campylobacter spp. ‡	350	11.4	[10.2-12.6]	20	8.8	[5.7-13.3]
	Rotavirus	1192	33.8	[32.2-35.5]	15	6.2	[3.7-10.1]
	Mixed infections excluded
	Putative EPEC*	289	7.5	[6.6-8.4]	10	4.3	[2.3-7.9]
	Salmonella spp.	184	4.8	[4.1-5.5]	15	6.7	[4.0-10.9]
	Shigella spp.	54	1.3	[1.0-1.7]	42	18.2	[13.6-23.8]
	Campylobacter spp. ‡	178	4.9	[4.2-5.7]	11	4.8	[2.7-8.5]
	Rotavirus	917	26.2	[24.7-27.8]	10	4.2	[2.2-7.7]

* Including all isolates identified as putative EPEC in Maradi ‡ Analysis restricted to July 2010-March 2012 doi:10.1371/journal.pone.0120275.t002 Fig 1. Distribution of organism identified in children with watery or bloody diarrhea by age group. doi:10.1371/journal.pone.0120275.g001

Table

3

. Demographic and clinical characteristics of children with watery or bloody diarrhea by agent identified from stools, after exclusion of mixed infections.

Table 5 .

 5 Salmonella serotypes identified in the study (N = 355).

	Subspecies	Serotypes	n	Subspecies	Serotypes	n
	I.	Aberdeen	2	I.	Mbao	5
	I.	Adelaide	5	I.	Menston	1
	I.	Agona	1	I.	Mesbit	1
	I.	Ank	2	I.	Molade	1
	I.	Antsalova	1	I.	Montevideo	4
	I.	Banana	11	I.	Muenchen	2
	I.	Belem	1	I.	Muenster	4
	I.	Brandenburg	1	I.	Ona	1
	I.	Carmel	2	I.	Oranienburg	4
	I.	Cerro	4	I.	Poona	6
	I.	Chester	3	I.	Rissen	1
	I.	Colindale	7	I.	Rubislaw	3
	I.	Corvallis	46	I.	Saintpaul	1
	I.	Denver	2	I.	Serenli	2
	I.	Derby	1	I.	Stanleyville	1
	I.	Ealing	1	I.	Suelldorf	1
	I.	Eastbourne	1	I.	Telelkebir	1
	I.	Elisabethville	1	I.	Tennessee	4
	I.	Enteritidis	56	I.	Tilene	3
	I.	Evry	1	I.	Typhi	2
	I.	Freetown	2	I.	Typhimurium	71
	I.	Gaminara	3	I.	Urbana	2
	I.	Give	8	I.	Vejle	7
	I.	Gombe	1	I.	Virchow	1
	I.	Hadar	1	I.	Waycross	1
	I.	Hato	2	I.	Wimborne	4
	I.	Havana	6	I.	Zinder	3
	I.	Herston	1	I.	Provisional new serotype I 44:z:z6	1
	I.	Hull	1	I.	New serotype Maradi (47:d:1,5)	2
	I.	Johannesburg	15	I.	1,3,19:d:-(monophasic)	3
	I.	Kalina	2	I.	4,5,12:-:1,2 (monophasic)	1
	I.	Kentucky	2	I.	4,5,12:i:-(monophasic)	4
	I.	Maastricht	12	IIIa.	41:z4,z23:-:-	1
	I.	Magherafelt	1

I., subspecies enterica; IIIa, subspecies arizonae; the 7 isolates (6 belonging to subsp. enterica and one to subsp. salamae) with a rough phenotype were not included. doi:10.1371/journal.pone.0120275.t005

Table 6 .

 6 Antibiotic resistance of EPEC, Salmonella spp. and Shigella spp. isolated in the study.

PLOS ONE | DOI:10.1371/journal.pone.0120275March 23, 2015

Acknowledgments

We would like to thank the children and their families for their participation in the study. We also thank the staff from the health centers and hospitals where the study was implemented in health districts of Madarounfa, Aguié, Maradi and Guidan Roumdji, Niger. We are also thankful to Abdoulaye Inoussa and Chou-ayb Yahaya from the Epicentre laboratory in Niger. We thank Isabelle Carle, Monique Lejay-Collin, Corinne Ruckly, Véronique Guibert, Marie Accou-Demartin, Laëtitia Fabre, Martine Garnier, Brigitte Chavinier-Jové, Chrystelle Roux, Erwan Trochu and Lucile Sontag for their excellent technical assistance.

Funding: Médecins Sans Frontières (MSF; www. msf.org) and Institut Pasteur funded this study. Epicentre receives core funding from Médecins sans Frontières. The French National Reference Center for E. coli, Shigella and Salmonella is cofunded by the Institut de Veille Sanitaire. The "Unité des Bactéries Pathogènes Entériques" belongs to the "Integrative Biology of Emerging Infectious Diseases" Laboratory of Excellence funded by the French Government "Investissement d'Avenir" programme (grant no.

Conclusions no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All relevant data are within the paper and its Supporting Information files.

Characterization of EPEC isolates

Of the 436 isolates initially screened as putative EPEC isolates by O serogrouping, a random sample of 74 isolates selected from rotavirus-negative children and all 37 isolates from rotavirus-positive children isolated in 2010 were characterized at the FNRC-ESS. Of these 111 isolates, 104 (93.7%) were confirmed as E. coli based on biochemical and phenotypic reactions, and 90/104 (86.5%) were confirmed to be EPEC due to the presence of the eae gene encoding intimin and the absence of other EHEC-associated virulence genes, stx1, stx2, and EHEC-hlyA (S2 Table ). Of the 90 EPEC, 88 (97.8%) were typical EPEC due to the presence of the bfpA gene carried by the EAF plasmid. Of the 14 other E. coli, 6 were actually EAEC due to the presence of the aggR gene and 8 did not contain any of the targeted virulence genes and were probably not pathogenic E. coli. The most frequent EPEC serogroups were O55 (32/90, 35.5%), O126 and O119 (13/90, 14.4% each) (S2 Table ). All but two EAEC also belonged to O55. Four of the 8 isolates without virulence genes belonged to serogroups common to EPEC such as O26, O114 and O126. There was no significant difference in the proportion of intimin-positive isolates among E. coli isolated in rotavirus-positive or negative children (p = 0.37).