Genomics of human and chicken Salmonella isolates in Senegal: Broilers as a source of antimicrobial resistance and potentially invasive nontyphoidal salmonellosis infections.

Salmonella enterica is the most common foodborne pathogen worldwide. It causes two types of diseases, a self-limiting gastroenteritis and an invasive, more threatening, infection. Salmonella gastroenteritis is caused by several serotypes and is common worldwide. In contrast, invasive salmonellosis is rare in high-income countries (HIC) while frequent in low- and middle-income countries (LMIC), especially in sub-Saharan Africa (sSA). Invasive Nontyphoidal Salmonella (iNTS), corresponding to serotypes other than Typhi and Paratyphi, have emerged in sSA and pose a significant risk to public health. We conducted a whole-genome sequence (WGS) analysis of 72 strains of Salmonella isolated from diarrheic human patients and chicken meat sold in multipurpose markets in Dakar, Senegal. Antimicrobial susceptibility testing combined with WGS data analysis revealed frequent resistance to fluoroquinolones and the sulfamethoxazole-trimethoprim combination that are among the most used treatments for invasive Salmonella. In contrast, resistance to the historical first-line drugs chloramphenicol and ampicillin, and to cephalosporins was rare. Antimicrobial resistance (AMR) was lower in clinical isolates compared to chicken strains pointing to the concern posed by the excessive use of antimicrobials in farming. Phylogenetic analysis suggested possible transmission of the emerging multidrug resistant (MDR) Kentucky ST198 and serotype Schwarzengrund from chicken to human. These results stress the need for active surveillance of Salmonella and AMR in order to address invasive salmonellosis caused by nontyphoidal Salmonella strains and other important bacterial diseases in sSA.

Antimicrobial Resistance Profile of Salmonella Isolates in Chicken Carcasses in Dakar, Senegal.

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.

Identification of Atypical El TorV. cholerae O1 Ogawa Hosting SXT Element in Senegal, Africa.

Vibrio cholerae O1 is the causative agent of cholera with classical and El Tor, two well-established biotypes. In last 20 years, hybrid strains of classical and El Tor and variant El Tor which carry classical ctxB have emerged worldwide. In 2004-2005, Senegal experienced major cholera epidemic with a number of cases totalling more than 31719 with approximately 458 fatal outcomes (CFR, 1.44%). In this retrospective study, fifty isolates out of a total of 403 V. cholerae biotype El Tor serovar Ogawa isolates from all areas in Senegal during the 2004-2005 cholera outbreak were randomly selected. Isolates were characterized using phenotypic and genotypic methods. The analysis of antibiotic resistance patterns revealed the predominance of the S-Su-TCY-Tsu phenotype (90% of isolates). The molecular characterization of antibiotic resistance revealed the presence of the SXT element, a self-transmissible chromosomally integrating element in all isolates. Most of V. cholerae isolates had an intact virulence cassette (86%) (ctx, zot, ace genes). All isolates tested gave amplification with primers for classical CT, and 10/50 (20%) of isolates carried classical and El Tor ctxB. The study reveals the presence of atypical V. cholerae O1 El Tor during cholera outbreak in Senegal in 2004-2005.

Evaluation of a new extraction protocol for yeast identification by mass spectrometry.

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.

First Description of the Extended Spectrum-Beta-Lactamase Gene blaCTX-M-109 in Salmonella Grumpensis Strains Isolated from Neonatal Nosocomial Infections in Dakar, Senegal.

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 ß-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.

High-quality draft genome sequence and description of Haemophilus massiliensis sp. nov.

Strain FF7(T) 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 FF7(T) 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 FF7(T) (= CSUR P859 = DSM 28247) as the type strain.

MALDI-TOF Mass Spectrometry: A Powerful Tool for Clinical Microbiology at Hôpital Principal de Dakar, Senegal (West Africa).

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.

Comparison of matrix-assisted laser desorption ionization-time of flight mass spectrometry and molecular biology techniques for identification of Culicoides (Diptera: ceratopogonidae) biting midges in senegal.

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.

Community-acquired diarrhea among children and adults in urban settings in Senegal: clinical, epidemiological and microbiological aspects.

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.