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Extra-intestinal pathogenic Escherichia coli (ExPEC), a predominant Gram-negative bacterial pathogen, express a wide range of virulence factors and is responsible of several diseases including urinary tract infections (UTI), nosocomial pneumonia, bacteremia, and neonatal meningitis. ExPEC isolates are often multidrug resistant (MDR) and clones producing extended-spectrum beta-lactamases (ESBL) are increasingly reported all over the world.Seventy-eight clinical ExPEC strains were selected for this study. The majority was from UTIs (n=51), while the rest (n=27) was from pus, sputum, bronchial fluid and vaginal samples (non-uropathogenic ExPEC). Interestingly, 49 out of the 78 ExPEC isolates where considered as community-acquired (CA) and 29 hospital-acquired (HA) bacteria. Antibiotic susceptibility testing was performed using the Kirby-Bauer disc diffusion method. Standard polymerase chain reaction (PCR) was used to screen major ESBL genes (blaCTX-M, blaOXA-1, blaTEM, blaSHV) and blaCTX-M variants (blaCTX-M-1, blaCTX-M-9, blaCTX-M-15, blaCTX-M-25).All the ExPEC strains were resistant to ampicillin, ticarcillin, amoxicillin/clavulanic acid combination, cefalotin, cefotaxime, ceftazidime, cefepime and aztreonam, but showed a high susceptibity to fosfomycin (98.7%, n = 77), ertapenem (96.2%, n = 75), and imipenem (100%). Moreover, isolates harbored at least one ESBL gene, including blaCTX-M (98.7%), blaOXA-1 (78.2%), blaTEM (44.9%) and blaSHV (3.8%). The CTX-M variants were also found with the predominance of blaCTX-M-1 (90.9%) and blaCTX-M-15 (90.9%) followed by blaCTX-M-9 (11.7%), while blaCTX-M-25 was not detected.Despite the resistance to most of the tested antibiotics, ExPEC isolates showed fortunately a good susceptibility to fosfomycin and carbapenems. blaCTX-M (blaCTX-M1, blaCTX-M15) and blaOXA-1 seem to be E. coli major ESBL genes circulating in Senegal. No significant difference was noted when comparing prevalence of ESBL genes detected from CA and HA strains, and from UPEC and non-uropathogenic ExPEC. The high level of resistance to antimicrobials observed stresses the need of establishing an epidemiological surveillance of antimicrobial resistance in both community and hospital settings.

Food-borne diseases represent a public major health problem, and drink-water, juice, meat, and milk products are usually involved. This study aimed to evaluate the antibiotic resistance of diarrheagenic E. coli isolated from dairy products consumed in Burkina Faso. Five hundred and twenty-two samples were gathered. Escherichia coli were isolated using Standard Microbiological Methods. A 16-plex polymerase chain reaction for virulence associated genes was applied. The standard disc diffusion methods were used to assess the susceptibility to 31 antibiotics. Classes 1, 2, 3 integrons were categorized using PCR. Results showed 1.92% (10/522) of milk products was contaminated by diarrheagenic E. coli. Enterotoxinogenic E. coli was found in 4.45% (4/89) of curds, 3.4% (3/88) of pasteurized milk, and 1.15% (1/87) of “degue”. Also, “degue” was contaminated at 2.3% (2/87) by atypical enteropathogenic E. coli. Antibiogram susceptibility showed that pathogenic isolated resists mainly to tetracycline, amoxicillin, ticarcillin, nalidixic acid, sulfonamide, and trimethoprim-sulfamethoxazole. Only the class 1 integrons was detected in 80% of diarrheagenic E. coli. Among this class 1 integrons, 4 strains contains a variable region, and the subsequent result showed a presence of dfrA7 gene coding for trimethoprim resistance. It appears in this study that dairy products are contaminated by enteropathogenic and enterotoxinogenic E. coli, which are resistant to antibiotics frequently used. This study therefore recommends the training of milk products transformers. Key words: Dairy products, diarrheagenic Escherichia coli, antibiotics resistance, integrons, Burkina Faso.

Background: In Burkina Faso, dirty water, in particular those of the stoppings and the gutter are used for irrigation of vegetables. The aim of this study is to contribute to the knowledge on the molecular level of Salmonella circulating in the hospitals and environment next to hospitals in Burkina Faso. Methods: Salmonella isolated from diarrhea stools between 2009 to 2015 and lettuce samples isolated in 2014 in Burkina Faso were characterized by simple Polymerase Chain Reaction using specific primers. Sequencing was performed using the BigDye terminator protocol on the ABI PRISM 310. The sequences obtained corresponding were searched using the BlastN program.Results: Out of 100 Salmonella isolated, 53% were from human and 47% from lettuce samples. Globally, the highest prevalence was observed with invA, misL, pipD, orfL and spvR genes in 97%, 96%; 74%; and 21%. Forty of these isolates carried class 1 integron, 31 from clinical samples and 9 from lettuce samples. Sequencing showed different gene with aadA1 in 13/15 strains, aadA7 and aac(3)-Id in 2/15 strains. Eight percent (8/100) of Salmonella harbored gyrB and parE genes. Sequencing showed no mutation in these genes. Three distinct Pulsed-Field Gel Electrophoresis (PFGE) types were observed from clinical samples with 90-95% similarity in each case. All Salmonella from lettuce had similar pulsotypes. Conclusion: This study showed that lettuce is a potential source of transmission of Salmonella causing diarrhea among human in Burkina Faso.

The incidence of the Salmonella contamination of poultry products in Senegal is unknown. Salmonella contamination and antimicrobial drug resistance profiles in chicken carcasses were investigated. Between July 2012 and July 2013, three types of chicken carcasses (broilers, laying hens, and premises chickens) obtained from retailers in the markets of Dakar and its suburbs were tested for Salmonella contamination. Salmonella strains were isolated from 300 chicken carcasses according to International Organization for Standardization ISO 6579 (2002) guidelines. In these samples, 273 isolates were obtained, belonging to 22 serovars, and 53% samples were contaminated with at least 1 serovar. Standardized techniques were used for the susceptibility testing and serotyping of isolates. Hygiene conditions, in terms of the cleanliness of stalls, the packing of chicken carcasses in bags, and the maintenance of the cold chain at the stall, were moderately poor. The three serovars most frequently identified were Salmonella Istanbul (28%), Salmonella Brancaster (19%), and Salmonella Kentucky (13%). Overall, 21% of isolates were resistant to quinolones and fluoroquinolones. Serovar Istanbul was resistant to tetracycline (TE) and trimethoprim + sulfamethoxazole (SXT). Serovars Brancaster and Kentucky were resistant to betalactams and to quinolones or fluoroquinolones. The uncommon serovar Senftenberg had the strongest resistance profile, displaying resistance to betalactams including imipenem (IMP). Large numbers of isolates were resistant to TE (66%) and SXT (47%). Resistance to cephalosporins (5%), chloramphenicol (2%), gentamicin (8%), and IMP (1%) was less frequent. A large proportion of the broilers sold in Dakar markets were contaminated with Salmonella. This situation probably resulted from poor hygiene conditions in chicken farms and slaughterhouses and from breaks in the cold chain at some point in the distribution of poultry products.

Strain FF9T was isolated in Dakar (Senegal) from a blood-culture taken from a 16-month-old child. MALDI-TOF analysis did not allow for identification. After sequencing, strain FF9T exhibited 98.18% similarity with the 16SrRNA sequence of Paenibacillus uliginis. A polyphasic study of phenotypic and genomic analyses showed that strain FF9T is Gram variable, catalase-positive, and presents a genome of 4,569,428 bp (one chromosome but no plasmid) with 4,427genes (4,352 protein-coding and 75 RNA genes (including 3 rRNA operons). The G+C content is 45.7%. On the basis of these genomic and phenotypic data analyses, we propose the creation of Paenibacillus dakarensis strain FF9T.

ObjectivesIn Burkina Faso (BF), dirty water, in particular those of the stoppings and the gutter are used for irrigation of vegetables. The aim of this study is to contribute to the knowledge on the molecular level of Salmonella Typhi and Paratyphi circulating in the hospitals and environment next to hospitals in BF.MethodsSalmonella Typhi and Paratyphi strains isolated from patients between 2009 to 2015 and lettuce samples isolated in 2014 in BF were characterized by simple PCR using specific primers.ResultsOut of 100 Salmonella isolated, 53% were from human and 47% from lettuce samples. Globally, the highest prevalence was observed with invA, misL, pipD, orfL and spvR genes in 97%, 96%; 74%; and 21%. Forty of these isolates carried class 1 integron, 31 from clinical samples and 9 from lettuce samples. Sequencing showed seven different gene cassette arrangements, with aadA1 in 13/15 strains, aadA7 and aac(3)-Id in 2/15 strains. Eight percent (8/100) of Salmonella harbored gyrB and parE genes with 6 from clinical and 2 from lettuce isolates. Sequencing showed no mutation in these genes. Three distinct PFGE types were observed from clinical samples with 90-95% similarity in each case. All Salmonella from lettuce had similar pulsotypes.ConclusionThis study showed the diversity virulence and resistance genes harbored of S. Typhi and Paratyphi from both clinical and lettuce samples in BF. Lettuce is a potential source of transmission of Salmonella causing diarrhea among human in BF.

Strain FF6T was isolated from the cervical abscess of a 4-year-old Senegalese boy, in Dakar, Senegal. MALDI-TOF MS did not provide any identification. This strain exhibited a 95.17% 16S rRNA sequence identity with Necropsobacter rosorum. Using a polyphasic study including phenotypic and genomic analyses, strain FF6T was an aero-anaerobic Gram-negative cocobacillus, oxidase positive, and exhibited a genome of 2,493,927 bp (1 chromosome but no plasmid) with a G+C content of 46.2% that coded 2,309 protein-coding and 53 RNA genes. On the basis of these data, we propose the creation of Necropsobacter massiliensis sp. nov.

Biting midges of the genus Culicoides are implicated as vectors for a wide variety of pathogens. The morphological identification of these arthropods may be difficult because of a lack of detailed investigation of taxonomy for this species in Africa. However, matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) profiling is efficient for arthropod identification at the species level. This study established a spectrum database of Culicoides spp. from Senegal using MALDI-TOF. Identification of Culicoides insects to the species level before mass spectrometry was performed on the basis of morphological characters. MALDI-TOF MS reference spectra were determined for 437 field-caught Culicoides of 10 species. The protein profiles of all tested Culicoides revealed several peaks with mass ranges of 2 to 20 kDa. In a validation study, 72 Culicoides specimens in the target species were correctly identified at the species level with a similarity of 95 to 99.9%. Four Culicoides protein profiles were misidentified. Nevertheless, six SuperSpectra ( C. imicola , C. enderleini , C. oxystoma , C. kingi , C. magnus , and C. fulvithorax ) were created. Abdomens of midges were used to amplify and sequence a portion of the mitochondrial cytochrome oxidase I gene (COI). The results obtained using the MALDI-TOF MS method were consistent with the morphological identification and similar to the genetic identification. Protein profiling using MALDI-TOF is an efficient approach for the identification of Culicoides spp., and it is economically advantageous for approaches that require detailed and quantitative information of vector species that are collected in field. The database of African Culicoides MS spectra created is the first database in Africa. The COI sequences of five Culicoides species that were previously noncharacterized using molecular methods were deposited in GenBank.

Until now, Planctomycetes bacteria were considered as environmental organisms. Nevertheless, some studies detected Planctomycetes DNA from human gut. We therefore explored the human gut Planctomycetes content. Planctomycetes-specific PCR primers were designed to amplify a 240-bp 16S rRNA gene fragment in human stool specimens from individuals in France and in Senegal and from endocarditis patients receiving antibiotics in France. PCR products were then cloned and sequenced. PCR detection revealed a significantly higher prevalence (1.8% vs 0.4%, p = 0.05) and higher diversity (62 vs 6 phylotypes, p = 0.02) of Planctomycetes 16S rRNA gene in stool specimens collected in Senegal than in France. Also, stool specimens from endocarditis patients exhibited non-significantly higher prevalence (0.6% vs 0.4%) and the ratio of phylotypes by positive patient (3 vs 1.5) than those collected from untreated French individuals. Gemmata sp. related sequences were found in 6/12 individuals. Planctomycetes organisms are a part of the human digestive tract microbiota. Their diversity varied by environment including the geographical origin of the individual and antibiotics treatment.

Diarrheal diseases remain a leading cause of death with 14.7 million deaths in 2001 and 26% of global mortality worldwide according to WHO. Shigella species are prevalent in tropical areas; they are present all the year, with epidemic outbreaks in rainy season. Between 2001 and 2010 one hundred ninety (190) strains of Shigella flexneri isolated from National Senegalese Enterobacteriaceae Center located at the Pasteur Institute in Dakar were studied. Susceptibility was performed by antibiogram following the CASFM recommendations. Detection and characterization of integrons and resistance genes was done by PCR using specific primers and sequencing. Antibiotic susceptibility showed high percentage resistance to tetracycline: 95%, cotrimoxazole 60%, ampicillin 55%. Nineteen strains were cephalosporin resistant (10%). Two isolates were resistant to quinolones and one was imipenem resistant. Genes tet, dfr, cat, bla tem1 , bla oxa30 , bla shv , bla CTX-M , blakpc,bla IMP , gyrA, gyrB, parC and parE were detected on isolates. Integrons harbored genes resistance. The class 1 integron predominated followed by class 2 integron. Genes bla oxa30 , aadA1/aadA2 dfrA1, dfrA7 were found on class 1 integron. Class 2 integron showed three different types cassettes. No class 3 integron was detected. Genes dfrA1, dfrA7, sat, and aadA1 were harbouring by integrons. Antibiotic susceptibility showed that Shigella flexneri strains are resistant to the first line drugs used to treat shigellosis in Senegal. Resistance to 3rd generation of cephalosporins and fluoroquinolones emerged and is of great concern. These molecules must be used with caution in the treatment of shigellosis.

Nosocomial infections are very common in African hospitals, particularly in neonatal units. These infections are most often caused by bacteria such as Escherichia coli, Klebsiella spp and Staphylococcus spp. Salmonella strains are rarely involved in nosocomial infections. Here, we report the first description of S. Grumpensis in neonatal infections in Senegal. Seventeen Salmonella strains were isolated from hospitalized infants' stool samples. The following resistance phenotype was described in strains: AMXRTICRCFR FOXRCFXRCTXRCAZRIMPSATMRNARNORRCIPRTMRGMRTERSXTR. All isolates were susceptible to imipenem, 15 out of 17 produced an extended spectrum s-lactamase (ESBL). blaOXA-1, blaSHV-1, blaTEM-1, blaCTX-M1 genes were detected in strains 8, 13, 5 and 8, respectively. blaCTX-M1 sequencing revealed the presence of blaCTX-M-109. Thirteen of the 17 Salmonella Grumpensis strains were analyzed by PFGE. These 13 isolates belonged to a single pulsotype and were genotypically identical. This is the first report of neonatal S. Grumpensis infections in Senegal, and the first report of blaCTX-M-109 in the genus Salmonella.

Strain FF7T was isolated from the peritoneal fluid of a 44-year-old woman who suffered from pelvic peritonitis. This strain exhibited a 16S rRNA sequence similarity of 94.8 % 16S rRNA sequence identity with Haemophilus parasuis, the phylogenetically closest species with a name with standing in nomenclature and a poor MALDI-TOF MS score (1.32 to 1.56) that does not allow any reliable identification. Using a polyphasic study made of phenotypic and genomic analyses, strain FF7T was a Gram-negative, facultatively anaerobic rod and member of the family Pasteurellaceae. It exhibited a genome of 2,442,548 bp long genome (one chromosome but no plasmid) contains 2,319 protein-coding and 67 RNA genes, including 6 rRNA operons. On the basis of these data, we propose the creation of Haemophilus massiliensis sp. nov. with strain FF7T (= CSUR P859 = DSM 28247) as the type strain. Electronic supplementary material The online version of this article (doi:10.1186/s40793-016-0150-1) contains supplementary material, which is available to authorized users.

Differential characteristics of Haemophilus massiliensis strain FF7T (data from this study), H. influenzae strain ATCC 33391T [1, 33], H. sputorum strain CCUG 13788T [38, 39], H. pittmaniae strain HK 85T [40, 41], Haemophilus felis strain TI189T [42, 43] and H. parasuis strain ATCC 19417T [33, 41]. na = data not available. (DOC 57 kb)

Antimicrobial susceptibility and minimum inhibitory concentrations (MIC) of Haemophilus massiliensis strain FF7T sp nov. (DOC 30 kb)

International audience; In this paper, we evaluate a rapid and safe pretreatment procedure using glass beads for MALDI-TOF yeast identification in a routine clinical laboratory avoiding the use of formic acid. We created a new yeast database library using 1186 yeasts, including 11 references strains. The database was tested using 2131 clinical isolates allowing accurate species-level identification in 98.9% (2107/2131) of cases with a score over 1.9 and in 99% (2123/2131) of the strains at the genus level. The new protocol is a rapid, reliable and safe procedure for the accurate identification of pathogenic Candida strains and requires minimal handling.

Reference mass spectrum from Haemophilus massiliensis strain FF7T. Spectra from 12 individual colonies were compared and a reference spectrum was generated. (DOCX 25 kb)

Gel view comparing Haemophilus massiliensis strain FF7T to the members of the Haemophilus genus. The gel view displays the raw spectra of all loaded spectrum files arranged in a pseudo-gel like look. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a Gray scale scheme code. The color bar and the right y-axis indicate the relation between the color a peak is displayed with and the peak intensity in arbitrary units. Displayed species are indicated on the left. (DOCX 192 kb)

Identification of fungal clinical isolates is essential for therapeutic management. In resource-limited settings, identification mostly relies on biochemical tests whose sensitivity and specificity are known to be insufficient for identification of closely related or newly described species. MALDI-TOF has been shown in favored countries to be a reliable and powerful tool for microorganism identification, including yeasts. The aim of this study was to compare MALDI-TOF with routine identification procedures in a resource-poor context. A total of 734 clinical specimens (502 vaginal swabs, 147 oral swabs, 61 bronchoalveolar lavage fluids and 24 stool samples) have been tested in the mycology unit of Fann Hospital, Dakar, Senegal. Strains isolated from culture were identified by both conventional phenotypic methods (germ tube formation and biochemical panels) and MALDI-TOF Saramis/VITEK MS, bioMerieux, France. In addition to comparing the final identification, we determined the time of obtaining the results and the cost for both approaches. Overall, 218 (29.7 %) samples were positive for Candida. MALDI-TOF MS enabled the identification of 214 of the 218 strains isolated (98.1 %) at species level. Phenotypic approach yielded identification for 208 strains (95.4 %). Congruence between the tests was observed for 203 isolates. A discrepancy was observed for one isolate identified as Candida krusei with the phenotypic approach and Candida tropicalis with the MALDI-TOF. In addition, ten isolates identified at genus level by phenotypic methods were identified as C. glabrata (n = 8), C. tropicalis (n = 1) and C. parapsilosis (n = 1) by MALDI-TOF. The turnaround time for identification was

Our team in Europe has developed the routine clinical laboratory identification of microorganisms by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). To evaluate the utility of MALDI-TOF MS in tropical Africa in collaboration with local teams, we installed an apparatus in the Hôpital Principal de Dakar (Senegal), performed routine identification of isolates, and confirmed or completed their identification in France. In the case of discordance or a lack of identification, molecular biology was performed. Overall, 153/191 (80.1%) and 174/191 (91.1%) isolates yielded an accurate and concordant identification for the species and genus, respectively, with the 2 different MALDI-TOF MSs in Dakar and Marseille. The 10 most common bacteria, representing 94.2% of all bacteria routinely identified in the laboratory in Dakar (Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, Enterobacter cloacae, Enterococcus faecalis, and Staphylococcus epidermidis) were accurately identified with the MALDI-TOF MS in Dakar. The most frequent misidentification in Dakar was at the species level for Achromobacter xylosoxidans, which was inaccurately identified as Achromobacter denitrificans, and the bacteria absent from the database, such as Exiguobacterium aurientacum or Kytococcus schroeteri, could not be identified. A few difficulties were observed with MALDI-TOF MS for Bacillus sp. or oral streptococci. 16S rRNA sequencing identified a novel bacterium, "Necropsobacter massiliensis." The robust identification of microorganisms by MALDI-TOF MS in Dakar and Marseille demonstrates that MALDI-TOF MS can be used as a first-line tool in clinical microbiology laboratories in tropical countries.

B. Sambe-Ba **, A. Seck , L.G. Timbine , A.A. Wane , A. Gassama-Sow * Unite de Bacteriologie Experimentale, Institut Pasteur de Dakar, 36 avenue Pasteur, BP 220 Dakar, Senegal b Laboratoire de Biologie Medicale, Institut Pasteur de Dakar, 36 avenue Pasteur, BP 220 Dakar, Senegal Unite de Bacteriologie Experimentale, Institut Pasteur de Dakar, 36 avenue Pasteur, BP 220 Dakar, Senegal

Background Only limited data are available relating to the etiology of diarrhea in children and adults in Senegal. The aim of this prospective study was to describe the epidemiology and etiology of community-acquired diarrheal infections in children and adults living in urban settings. Methods A prospective study was carried out from March 2009 to December 2010, in the urban region of Dakar, Senegal. Patients with acute diarrhea were enrolled, interviewed to collect their clinical history, and their stools were tested for bacteria, virus and parasites. Results A total of 223 patients (including 112 children younger than five years old) with diarrhea were included. At least one enteropathogen was detected in 81% (180/223) of the patients: 29% (64/223) had bacterial infections (mainly diarrheagenic E. coli and Shigella spp), 21% (39/185) viral infections (mainly rotavirus) and 14% (31/223) parasitic infections. Co-infection was identified in 17.8% (32/180) of the patients. Viral infection was significantly more frequent in children under five years old during the dry season. Bacteria and parasites were equally frequent in all age groups. There was a seasonal variation of bacterial infections during the study period, with a higher proportion of infections being bacterial, and due to Salmonella spp. in particular, during the rainy season. Conclusion Our study suggests that in urban settings in Senegal, rotavirus is the principal cause of pediatric diarrhea during the dry season and that the proportion of bacterial infections seems to be higher during the rainy season. Further work is needed to document the burden of diarrheal diseases in sub-Saharan urban communities and to identify risk factors, including those linked to the rapid and unplanned urbanization in Africa.