Nathalie Guessennd

Population structure of clinical Pseudomonas aeruginosa from West and Central African countries.

Background Pseudomonas aeruginosa (PA) has a non-clonal, epidemic population with a few widely distributed and frequently encountered sequence types (STs) called ‘high-risk clusters’. Clinical P. aeruginosa (clinPA) has been studied in all inhabited continents excepted in Africa, where a very few isolates have been analyzed. Here, we characterized a collection of clinPA isolates from four countries of West and Central Africa. Methodology 184 non-redundant isolates of clinPA from hospitals of Senegal, Ivory Coast, Nigeria, and Central African Republic were genotyped by MLST. We assessed their resistance level to antibiotics by agar diffusion and identified the extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) by sequencing. The population structure of the species was determined by a nucleotide-based analysis of the entire PA MLST database and further localized on the phylogenetic tree (i) the sequence types (STs) of the present collection, (ii) the STs by continents, (iii) ESBL- and MBL-producing STs from the MLST database. Principal Findings We found 80 distinct STs, of which 24 had no relationship with any known STs. ‘High-risk’ international clonal complexes (CC155, CC244, CC235) were frequently found in West and Central Africa. The five VIM-2-producing isolates belonged to CC233 and CC244. GES-1 and GES-9 enzymes were produced by one CC235 and one ST1469 isolate, respectively. We showed the spread of ‘high-risk’ international clonal complexes, often described as multidrug-resistant on other continents, with a fully susceptible phenotype. The MBL- and ESBL-producing STs were scattered throughout the phylogenetic tree and our data suggest a poor association between a continent and a specific phylogroup. Conclusions ESBL- and MBL-encoding genes are borne by both successful international clonal complexes and distinct local STs in clinPA of West and Central Africa. Furthermore, our data suggest that the spread of a ST could be either due to its antibiotic resistance or to features independent from the resistance to antibiotics.

Outbreak of metallo-β-lactamase VIM-2-positive strains of Pseudomonas aeruginosa in the Ivory Coast

Sir, Acquired metallo-b-lactamases (MBLs) are increasingly recognized as important determinants ofb-lactam resistance in Pseudomonas aeruginosa. To date, seven types of MBL enzyme (IMP, VIM, NDM, SPM, GIM, AIM and FIM) have been identified in this opportunistic pathogen. Since its first description in France in 1996, the epidemiologically successful enzyme VIM-2 has been traced in resistant P. aeruginosa strains from various European countries (e.g. Spain, Italy, Croatia, Greece, the Netherlands and Turkey) and more recently in strains from northern (Tunisia and Algeria) and southern (South Africa) Africa. – 6 One study also mentioned its occurrence in Kenya. Illustrating the spread of the VIM-2-encoding gene, blaVIM-2, in sub-Saharan Africa countries, we found here that P. aeruginosa strains from the Ivory Coast harboured an atypical class I integron previously described in sporadic isolates from other continents. Twelve P. aeruginosa isolates that were non-susceptible to imipenem according to the EUCAST interpretation rules (imipenem MIC .4 mg/L) were recovered from separate patients hospitalized in the intensive care unit of the teaching hospital in Abidjan, Ivory Coast, between February 2009 and November 2011. A double-disc (10 mg of imipenem, 2.5 mmol of EDTA) synergy test and an MBL Etest (imipenem+EDTA; bioMerieux, Craponne, France) were performed and found to be positive with seven of these isolates, suggesting the production of an Ambler class B b-lactamase (MIC of imipenem ≥256 mg/L; MIC of imipenem+EDTA 32 mg/L). PCR and DNA sequencing experiments revealed the presence of the gene blaVIM-2 in these isolates. All were highly resistant to carbapenems (imipenem MIC ≥128 mg/L, meropenem MIC ≥32 mg/L and doripenem MIC ≥32 mg/L), ceftazidime (MIC ≥64 mg/L), aminoglycosides (tobramycin MIC ≥128 mg/L) and ciprofloxacin (MIC ≥16 mg/L), but remained susceptible to aztreonam (MIC ≤1 mg/L) and colistin (MIC ≤4 mg/L). Six isolates were responsible for bloodstream (n1⁄43), urinary tract (n1⁄42) and pulmonary (n1⁄41) infections; the last one was considered to be a cathetercontaminant. Theirgenetic relatednesswas investigatedbymultiple-locusvariablenumber tandem-repeat analysis using eight variable-number tandem repeats, as described previously. All the VIM-2-positive isolates turned out to belong to the same epidemic clone (serotype O6), which was assigned to a new sequence type (ST), ST1488, after multilocus sequence typing (MLST) analysis. Interestingly, the closest ST found in the PubMLST database (http://pubmlst.org/paeruginosa), ST233, relates to a P. aeruginosa strain (serotype O6) identified in Sweden in 2006 from a patient repatriated from Ghana. Nucleotide sequence alignments demonstrated that this strain isolated from Ghana carried the same blaVIM-2-containing integron, In559, as ST1488 (Figure 1). Since Ghana and the Ivory Coast are neighbouring countries, this suggests that closely related multidrug-resistant genotypes are present in this region of the African continent. Screening for additional b-lactamase genes by PCR (blaTEM, blaSHV, blaPER, blaGES, blaVEB, blaIMP, blaNDM, blaSPM, blaPSE and blaOXA including blaOXA-198) revealed that the seven isolates except one (12.871) harboured blaOXA-4, which codes for the narrow-spectrum oxacillinase OXA-4. The gene was located on a class 1 integron upstream of the aadA2 and cmlA1 cassettes, which code for the aminoglycoside-modifying enzyme ANT(3′′) (streptomycin and spectinomycin resistance) and for an active efflux pump accommodating chloramphenicol, respectively. PCR experiments using primers specific to 5′ and 3′ conserved sequences (5′CS and 3′CS) failed to detect a class 1 integron associated with blaVIM-2 in the strains. To characterize the genetic environment of blaVIM-2, a BamHI library of strain 12.871 DNA was cloned into the BamHIrestricted plasmid pK18 (Km). Transformants of E. coli DH5a were selected on Mueller–Hinton agar plates containing 100 mg/L ampicillin and 30 mg/L kanamycin. Compared with strain DH5a, carrying the empty pK18 vector, all the recombinant clones tested displayed high levels of resistance to b-lactams except aztreonam. Sequence analysis of the 6224 kb insert from one of these clones led to the identification of a class 1 integron with a 5′CS sequence and gene tniCat its 3′ end. This genetic structure wasfound to carry the following gene cassettes downstream of the integrase gene intI1: aacA7 (encoding acetyltransferase AAC6′-Il, which confers amikacin resistance), blaVIM-2, dhfrB5 (encoding a dihydrofolate reductase, providing trimethoprim resistance) and aacC5b (encoding an uncharacterized aminoglycoside acetyltransferase). Interestingly, this genetic structure was identical to that of blaVIM-2-containing integrons characterized in P. aeruginosa from the USA (AY943084), Taiwan, Russia (AM749810), India (HQ005291) and the Indian Ocean (AM296017), but was different from integrons reported so far in African countries other than Ghana (Figure 1). Interestingly, upstream of the intI1 gene, we could identify the transposase gene tnpA from ISPa58 (ISfinder database, http://www-is.biotoul.fr). The sequence was 100% identical to that of tnpA belonging to the Tn3/ Tn21 family (JX194160). Repeated assays to visualize a plasmid by

Epidemiology and antibiotic resistance phenotypes of diarrheagenic Escherichia coli responsible for infantile gastroenteritis in Ouagadougou, Burkina Faso

The emergence and persistence of multidrug-resistant (MDR) diarrheagenic Escherichia coli (DEC) causing acute diarrhea is a major public health challenge in developing countries. The aim of this study was to evaluate the resistance phenotypes of DEC isolated from stool samples collected from children less than 5 years of age with acute diarrhea living in Ouagadougou/Burkina Faso. From August 2013 to October 2015, this study was carried out on 31 DEC strains of our study conducted in “Centre Médical avec Antenne Chirurgicale (CMA)” Paul VI and CMA of Schiphra. DEC were isolated and identified by standard microbiological methods and polymerase chain reaction (PCR) method was used to further characterize them. Antimicrobial susceptibility testing was done based on the disk diffusion method. DEC isolates were high resistant to tetracycline (83.9%), amoxicillin (77.4%), amoxicillin clavulanic acid (77.4%), piperacillin (64.5%), and colistin sulfate (61.3%). The most resistant phenotype represented was the extended spectrum β-lactamase (ESBL) phenotype (67.7%). Aminoglycosides were 100% active on enteroinvasive E. coli (EIEC) and enterohemorrhagic E. coli (EHEC). All the DEC isolates exhibited absolute (100%) sensitivity to ciprofloxacin. Monitoring and studying the resistance profile of DEC to antibiotics are necessary to guide probabilistic antibiotic therapy, especially in pediatric patients.

Characterization of virulence potential of Pseudomonas aeruginosa isolated from bovine meat, fresh fish, and smoked fish

Pseudomonas aeruginosa owns a variability of virulence factors. These factors can increase bacterial pathogenicity and infection severity. Despite the importance of knowledge about them, these factors are not more characterized at level of strains derived from local food products. This study aimed to characterize the virulence potential of P. aeruginosa isolated from various animal products. Several structural and virulence genes of P. aeruginosa including lasB, exoS, algD, plcH, pilB, exoU, and nan1 were detected by polymerase chain reaction (PCR) on 204 strains of P. aeruginosa. They were isolated from bovine meat (122), fresh fish (49), and smoked fish (33). The 16S rRNA gene was detected on 91.1% of the presumptive strains as Pseudomonas. The rpoB gene showed that 99.5% of the strains were P. aeruginosa. The lasB gene (89.2%) was the most frequently detected (p < 0.05). In decreasing importance order, exoS (86.8%), algD (72.1%), plcH (72.1%), pilB (40.2%), and exoU (2.5%) were detected. The lasB gene was detected in all strains of P. aeruginosa serogroups O11 and O16. The prevalence of algD, exoS, and exoU genes in these strains varied from 51.2% to 87.4%. The simultaneous determination of serogroups and virulence factors is of interest for the efficacy of surveillance of infections associated with P. aeruginosa.

Comparative study of the impact of the administration of Amoxicillin and Algo-Bio® alternative substance to antibiotics, on the level of selection of resistant Enterobacteriaceae in the digestive flora of piglets

OBJECTIVES The aim of study was to evaluate by comparative study the level of selection of antibiotic-resistant Enterobacteriaceae in the digestive microbiota of piglets when using amoxicillin and Algo-Bio®. METHODS Amoxicillin and Algo-Bio® administration was carried out over a period of 5 days (D0-D4) at a dose of 1mL/10kg body weight. A phenotypic study was carried out with enumeration of resistant Enterobacteriaceae on MacConkey agar plates in the presence and absence of amoxicillin. Escherichia coli isolates were identified and were subjected to antimicrobial susceptibility testing. RESULTS The percentages of amoxicillin-resistant Enterobacteriaceae before treatment ranged from 10-15% for the four groups of piglets. Following treatment initiation, on the second day (D1) to the fifth day (D4) of treatment, the percentages increased to 54-87% for the groups treated with amoxicillin. In the group treated with Algo-Bio® and the controls, the percentages were <50%. The percentage of amoxicillin-resistant E. coli strains to the associated antibiotics increased during days of amoxicillin treatment, whereas in the control and Algo-Bio® groups the percentages of E. coli resistant to antibiotics did not increase. CONCLUSION The results indicated that Algo-Bio® constitutes a good alternative prophylactic to antibiotics to reduce bacterial growth in the digestive tract of animals.

Distribution of resistance genes encoding ESBLs in Enterobacteriaceae isolated from biological samples in health centers in Ouagadougou, Burkina Faso

ObjectiveResistance to antibiotics most especially third generation cephalosporins has assumed a worrisome dimension globally. Genes conferring these resistance which are mediated by enzymes known as extended spectrum beta-lactamases (ESBLs) are now wide spread among several Enterobacteriaceae species. However there is paucity of data regarding the distribution of these genes in Burkina Faso. Hence this prospective study aims to determine the prevalence and distribution of ESBL encoding genes in ESBL producing Enterobacteriaceae strains isolated from clinical samples of patients attending the three major hospitals in Ouagadougou Burkina Faso.ResultsESBL-encoding genes were assayed in 187 ESBL producing Enterobacteriaceae strains. Among these isolates, the prevalence of ESBL-producing strains with blaTEM, blaSHV and blaCTX-M genes were 26.2% (49/187), 5.9% (11/187) and 40.1% (75/187) respectively. The association of ESBL encoding genes with health centers was statistically significant (p = 0.0209). Approximately 39.6% of E. coli harbored CTX-M and Klebsiella spp. 5.9%. This study demonstrates the dissemination of TEM, SHV and CTX-M genes in ESBL producing Enterobacteriaceae strains in Ouagadougou. Continuous spread of these bacteria poses great public health risk, thus increased surveillance and regulation of antibiotics use is imperative in Burkina Faso.

Molecular characterization of diarrheagenic Escherichia coli in children less than 5 years of age with diarrhea in Ouagadougou, Burkina Faso

Diarrheagenic Escherichia coli (DEC) is important bacteria of children’s endemic and epidemic diarrhea worldwide. The aim of this study was to determine the prevalence of DEC isolated from stool samples collected from children with acute diarrhea living in Ouagadougou, Burkina Faso. From August 2013 to October 2015, stool samples were collected from 315 children under 5 years of age suffering from diarrhea in the “Centre Médical avec Antenne Chirurgicale (CMA)” Paul VI and the CMA of Schiphra. E. coli were isolated and identified by standard microbiological methods, and the 16-plex PCR method was used to further characterize them. Four hundred and nineteen (419) E. coli strains were characterized, of which 31 (7.4%) DEC pathotypes were identified and classified in five E. coli pathotypes: 15 enteroaggregative E. coli (EAEC) (48.4%), 8 enteropathogenic E. coli (EPEC) (25.8%) with 4 typical EPEC and 4 atypical EPEC, 4 enteroinvasive E. coli (EIEC) (12.9%), 3 enterohemorrhagic E. coli (EHEC) 9.67%, and 1 enterotoxigenic E. coli (ETEC) 3.2%. The use of multiplex PCR as a routine in clinical laboratory for the detection of DEC would be a useful mean for a rapid management of an acute diarrhea in children.

Antimicrobial susceptibility of extended-spectrum beta-lactamase producing Enterobacteriaceae causing urinary tract infections in Ouagadougou, Burkina Faso

Objective: To determine the frequency of extended-spectrum beta lactamase producing Enterobacteriaceae (ESBL) and other antibioticsresistant bacteria in urinary tract isolates. Study Design: prospective and experimental study. Methodology: Place and duration of study :YalgadoOuedraogo University Hospital Center, Charles De Gaulle Pediatric Hospital Center, Saint Camille Hospital and National Public Health Laboratory, Ouagadougou, from November 2014 to October 2015. All Enterobacteriaceae strains isolated from urinary samples of patients were identifiedusing API 20E chemical gallery (BioMerieux, France). All strains were subjected to an array of 14 antibiotics to study their drug susceptibility by using Kirby- Baeurdisk diffusion method. Detection of ESBL was carried out by double disk diffusion technique. Statistical analysis was performed by Microsoft Excel and Anova one-way GrapPad Prism version 5.01. Chi-square (χ 2 ) test was used to determine significance. A p ˂ 0.05was considered to be statistically significant. Results: A total of 324 isolates of Enterobacteriaceae were identified during the study period, including211(65%) E. coli , 75 (23%) Klebsiella spp., 18 (6%) Enterobacter spp., 11 (3%) Proteus spp., 5 (2%) Citrobacter spp., Serratia spp. 3 (1%).All the clinical isolates were susceptible to imipenem. Resistance to amikacinwas 14% (45/324); gentamicin 54% (175/324); tobramycin 58% (187/324); nalidixic acid 72% (234/324),ciprofloxacin 63% (204/324) and to cotrimoxazole 83% (269/324).The overall rate of the EBSL producing strains was 35% (114/324). Their susceptibility to antibiotics was (imipenem,amikacin, cefoxitin and fosfomycin) 100% (114/114), 93% (106/114), 74% (84/114) and 84% (96/114) respectively. ESBL positivity within individual organism group was highest in Escherichia coli 64% (73/324) followed by Klebsiella spp. 28% (32/324), Enterobacter spp. 3% (4/324), Proteus spp. and Citrobacter spp. 2% (2/324). Conclusion: The results showeda high frequency of ESBL producing Enterobacteriaceae , especially Escherichia coli and Klebsiella spp. The data points to theneed of routine detection and surveillance of ESBL producing bacteria in Burkina Faso. Keywords: Antimicrobial susceptibility, Enterobacteriaceae , Urine, Burkina Faso

Comparative Evaluation of Molecular Detection Performance of Pseudomonasaeruginosa based on Phylogenetic Markers 16S RNAr, recA, rpoB and ITS1

P. aeruginosa may be involved in the poisoning of food. It is highly pathogenic to immunocompromised subjects or weakened, causing a high rate of morbidity and mortality. The aim of this study was to determine the phylogenetic marker suitable for molecular identification of Pseudomonas aeruginosa. The purity and concentration of the nucleic acids were determined by spectrophotometry. Sensitivity reactions using phylogenetic markers (16S RNAr, recA, rpoB, STS1) and the threshold detection of 42 strains were assessed by polymerase chain reaction (PCR). With an average absorption at 230 nm of 2.1, the DNA extracts has an average ratio (A260/A280) of 1.7. The threshold detection of Pseudomonas aeruginosa reference strain ATCC 27853 was 0.8 μg/ml for rpoB and 7.6 μg/ml for each of 16S markers RNAr and recA. The threshold detection of positive control strains CP2: 1125A and CP3: API was 1.2 μg/ml and 0.1 μg/ml for using rpoB gene, respectively. This threshold was respectively 12.3 μg/ml and 0.9 μg/ml for the recA gene. The sensitivity of the rpoB housekeeping gene was 97.4% followed by the recA and 16S RNAr with 87.2% and 82.1%, respectively. The phylogenetic resolution of the rpoB genes was higher than that of the 16S rRNA and recA genes. No sensitivity reaction was observed with ITS1 marker. The quality, purity of the nucleic acids and the choice of phylogenetic marker are among the most critical factors for PCR analysis.