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The burden of anaerobic bacteremia ranged from 2 to 14% of patients with positive BCs among centers, reflecting different hospital sizes and medical activities. Cutibacterium acnes was the most frequently recovered anaerobe in some centers, suggesting different medical activities / BCs laboratory procedures. Antimicrobial resistance rate in the B. fragilis group is worrisome. It may be associated with treatment failures and requires continuous surveillance.

The burden and epidemiology of anaerobic bacteremia is poorly described. The aim of the study was to characterize the incidence and evolution trends of anaerobic bacteremia in Europe and neighbouring countries in 2012 and 2018 both in terms of microbial epidemiology and antimicrobial resistance. Retrospective multicenter study: 17 laboratory hospitals from 12 countries Results data: total number of adult patients with anaerobes, patients’ age, gender and hospitalization ward, laboratory methods, identification of isolates and antimicrobial susceptibility results were analysed. Burden of anaerobic bacteremia • The mean frequency of patients with anaerobes in BCs was 5% of patients, with important variations according to centers (2% to 14%), probably reflecting their medical activity. The reported incidence moderately increased between 2012 and 2018. • These patients were frequently hospitalized in emergency wards and intensive care units (25% and 22% of isolates), followed by medicine, digestive surgery and oncology (6.7%) Epidemiology of anaerobes in blood cultures • 50% of anaerobic BCs were obtained from emergency and Intensive care units, demonstrating their clinical importance • 4 genera represented 75% of isolates : Bacteroides, Cutibacterium, Clostridium and Fusobacterium • Cutibacterium acnes frequency significantly varied among centers, suggesting variations in Laboratory management of BCs (incubation time). Antimicrobial resistance reported rates • Antimicrobial resistance rates varied according to genera and remained stable between 2012 and 2018. • Clindamycin resistance was the most frequently observed phenotype, predominating in Bacteroides spp, Clostridium spp and anaerobic Gram positive rods. • 10% of Bacteroides spp were resistant to piperacillin- tazobactam. Carbapenems and metronidazole resistance rate were below 5%. • Metronidazole resistance was frequently reported in anaerobic Gram Positive cocci

International audience

International audience; Objectives: On the last decade, ECC members has been defined by Hoffmann and Roggenkamp in 13 genetic clusters (I to XIII) based most exhaustively on hsp60 sequencing. More recently, Chavda et al. in 2016 proposed a new phylogenic classification by using both average nucleotide identity and a single-nucleotide polymorphism based phylogeny. The aim of this study is to improve the determination method based on hsp60.Methods: All the available ECC genomes (1.324) in the patric database v3.5.27 have been downloaded. The correct specie identification and the absence of contaminant specie were checked using the rMLST data base provided by PubMLST. The hsp60, rpoB and ampC genes were extract form all the genomes and clustered using cd-hit with an 99% identical cut off. A distance matrix between all the genomes based on the Kmer content was determined using Mash software and a neighbor joining tree (NJT) was constructed using the neighbor software available in the PHYLIP package. The tree and the molecular typing of all the strains were represented using itol.Results: Using Mash allowed to obtain a distance matrix of more than a thousand genomes in less than one hour. Clusters recovered in the NJT were in ad equation with the hsp60 clustering method for C11, C2, C5, C7. Nevertheless, genomes which belonged to the same clusters such as C9 or C4 appeared to be very divergent to each other. Furthermore, genomes which belonged to C13 seemed to be miss-classified C6. As the consequence, the hsp60 method could be not enough discriminant and precise to allow a correct clusterization and can lead to typing errors.The diversity of the rpoB gene sequences didn’t explained the phylogenetic clusters observed. Especially, it appeared that, C8 and C6 clusters shared the same rpoB sequences, as the consequence of what, rpoB sequences can’t be used as a clustering method. Interestingly, the diversity of the blaACT sequences improved the discrimination between the isolates and avoided mis-clusterization.Conclusion: The findings in this study provide an additional approach to identify in a more precise way and easily the cluster to which belongs the isolate of ECC.

International audience; Objectives: Members of the Enterobacter cloacae complex (ECC) are major opportunistic pathogens, especially among intensive care unit (ICU) patients. The aim of this study was to decipher the phenotypic and genomic evolution of an ECC clone that acquired a multidrug resistance phenotype in an ICU patient. Methods: During a four-month period, 8 MDR ECC strains were recovered from the same patient: 5 from clinical samples and 3 from rectal swabs. The patient benefited from an escalation of antibiotic treatment until he died. MIC values of 18 antibiotics were determined by the broth microdilution method. Whole-genome sequencing was performed for the 8 strains and a Single-Nucleotide Polymorphism (SNP) analysis was performed after alignment against the ECC reference genome (ATCC 13047). Results: In strains isolated from the 3rd month, a significant increase in MICs (at least 4 fold change) was observed for ceftazidime, cefepime, temocillin, ertapenem, tigecycline and chloramphenicol. All strains harbored the same antibiotic resistance genes against β-lactams, aminoglycosides, fluoroquinolones, sulphonamides, trimethoprime and chloramphenicol. The 8 MDR ECC belonged to the same cluster 6 (ECC6) and were genetically related. No genetic evolution was found in chromosomal genes usually involved in β-lactam resistance (i.e. ampD, ampR, nagZ, dacA, dacB, acrAB-tolC, ompK35, ompK36). The isolates differed by 5 SNPs located in metabolism genes and a deletion of 13 bases in romR, which encodes for TetR-type transcriptonal regulator. This latter is known to play a repressor role on the expression of porins such as ompK35, whose the downregulation might be involved in MIC increases observed here. Conclusion: Consequent and long-term antibiotic treatment in one patient indicated the selection of a unique MDR ECC6 population where antibiotic resistance evolved more by chromosomal modifications than acquired antibiotic resistance genes. The analysis of the different genetic features through all the 8 MDR ECC6 strains allowed us to highlight a candidate gene that could be implicated in carbapenem, chloramphenicol and tigecyclin resistance. Further phenotypic and molecular characterization by site-directed mutagenesis is in progress.

International audience; Introduction :Escherichia coli représente 62% des entérobactéries sécrétrices de béta-lactamase à spectre étendu (BLSE) en France. Une surveillance génomique a été initiée sur les E. coli sécrétrices de béta-lactamase au CHU de Caen depuis 2012. Au cours de cette étude, deux épidémies d’E. coli sécrétrices de BLSE au sein de services de réanimation néonatale a été particulièrement investiguée.Matériels et méthodes :Entre Juin et Aout 2017, 28 souches d’E. coli sécrétrices de BLSE isolées dans les services de réanimation néonatales des hôpitaux de Cherbourg et de Caen ont été collectées. Au total 94 souches ont été séquencées dans le cadre de la surveillance. Les caractéristiques des bactéries : Sequence-Type (ST), sérotype, gènes de résistance, de virulence et plasmides ont été déterminés in silico grâce à différentes bases de données. Une phylogènie basée sur la recherche de Single Nucleotide Polymorphism (SNP) a été réalisée par mapping sur un génome de référence puis par comparaison à l’ensemble des 3765 génomes d’E. coli ST131 disponibles dans les bases de données.Résultats :Douze patients âgés d’un à cinq mois ont été colonisés ou infectés par une souche d’E. coli sécrétrice de BLSE entre Juin et Aout 2017. Les bactéries ont été isolées à partir de selles (n=22), yeux (n=3), hémocultures (n=2) et tractus respiratoire (n=1). Toutes les souches des services de réanimation appartenaient au clade ST131 O16:H5 Fim 41 sécrétant la béta-lactamase CTX-M-27 confirmant le caractère épidémique de cet évènement au vu des caractéristiques différentes des E. coli circulant au CHU de Caen depuis 2012. De plus, la phylogénie basée sur les SNP indiquait une faible divergence entre ces souches avec un nombre moyen en SNP de 77 alors qu’elles divergeaient des génomes d’E. coli BLSE ST131 non épidémique de plus de 13000 SNP. L’augmentation de la puissance des analyses par ajout de 3765 génomes d’E. coli ST131, a permis (i) de révéler que les 2 épidémies de Cherbourg et Caen étaient indépendantes (900 SNP de différence) et (ii) de démontrer une récente dissémination mondiale du clade E. coli ST131 O16:H5.Conclusion :La béta-lactamase CTX-M-27 dissémine rapidement au sein d’hôpitaux Français via un clade émergent d’E. coli ST131 O16:H5 Fim 41 différent des clades précédemment décrits. Des études phénotypiques doivent être menées afin de comprendre le succès de ce clone.

International audience; Introduction :Les entérobactéries sécrétrices de béta-lactamase à spectre étendu (BLSE) sont responsables d’une augmentation de la morbi-mortalité infectieuse. Cette étude s’est intéressée à la diversité génomique des souches d’E. coli sécrétrices de BLSE au de CHU de Caen sur une période de 5 ans.Matériels et méthodes :Cinquante-trois souches représentant la première indentification d’une BLSE chez des patients uniques, isolées entre 2012 et 2016, à partir d’écouvillonnages rectaux ont été séquencées en génome entier. Les caractéristiques des bactéries : Sequence-Type (ST), sérotype, gènes de résistance et gènes de virulence ont été déterminés in silico grâce aux bases de données.Résultats :Les prélèvements provenaient de 16 différents services, majoritairement les services de médecine, de chirurgie puis de réanimation (40%, 31%, 29%). Le sex ratio H/F était de 0,9. L’âge moyen des patients était de 64 ans ; 55% avaient été hospitalisés dans les 3 mois précédents ; 51% ont reçu une antibiothérapie au cours de leur hospitalisation et 14% avaient voyagé hors de France.Au total, 34 différents ST et 35 sérotypes ont été retrouvés. Les populations E. coli les plus fréquentes étaient les ST131 O25:H4 (n=6), ST10 O89:H9 (n=3) et ST162:O9:H9 (n=2). Onze différentes béta-lactamases ont été détectées, les plus fréquentes étaient : CTX-M-1 (n=18), CTX-M-14 (n=9), CTX-M-15 (n=7). Pour 27 patients (49%), la souche d’E. coli BLSE a été identifiée avant 48h d’hospitalisation, principalement CTX-M-14. Tandis que l’enzyme « nosocomiale » était volontiers CTX-M-15 (délai moyen d’acquisition 19 jours, min/max : 0/66 jours). Enfin, CTX-M-14 et SHV-12 étaient significativement plus retrouvées pour les souches d’E. coli circulantes au sein des services de réanimation (p=0,02, p=0,02) supposant une circulation diffuse de souches épidémiques.Conclusion :Cette étude d’épidémiologie génomique a permis de révéler une grande diversité de populations des souches d’E. coli sécrétrices de BLSE au CHU de Caen depuis 2012. Cela supporte que dans 50% des cas le portage de ces bactéries était probablement d’origine communautaire (détection dans les 48h après l’admission). D’autres souches hospitalières, communautaires et vétérinaires sont en cours de séquençage afin d’augmenter la puissance de l’étude sur leurs attributions et leurs circulations en Normandie qui semblent assez étendues.

The environmental bacterium $\textit{Burkholderia pseudomallei}$ causes an estimated 165,000 cases of human melioidosis per year worldwide and is also classified as a biothreat agent. We used whole genome sequences of 469 $\textit{B. pseudomallei}$isolates from 30 countries collected over 79 years to explore its geographic transmission. Our data point to Australia as an early reservoir, with transmission to Southeast Asia followed by onward transmission to South Asia and East Asia. Repeated reintroductions were observed within the Malay Peninsula and between countries bordered by the Mekong River. Our data support an African origin of the Central and South American isolates with introduction of B. pseudomallei into the Americas between 1650 and 1850, providing a temporal link with the slave trade. We also identified geographically distinct genes/variants in Australasian or Southeast Asian isolates alone, with virulence-associated genes being among those over-represented. This provides a potential explanation for clinical manifestations of melioidosis that are geographically restricted.

National audience; Background. Efflux is a mechanism that has been previously reported to increase fluoroquinolone (FQ) resistance levels when it is upregulated in clinical isolates of Salmonella enterica mainly of serotype Typhimurium (1). In this study efflux related genes were investigated in the emerging FQ-resistant epidemic S. enterica serotype Kentucky ST198 clone (2) for the presence of mutations in their regulatory genes, and for their expression levels. Methods. Among a representative panel of thirty serotype Kentucky strains from Egypt or east Africa with decreased FQ susceptibility, three strains overproducing the AcrAB-TolC efflux system were detected and studied (3). Two FQ-resistant strains with substitutions in QRDR and with basal expression level of AcrABTolC and the susceptible reference strain 98K were used as control. Genetic relatedness was determined by XbaI-pulsed field gel electrophoresis and multilocus sequence typing. Presence of mutations in the ramRA, soxRS, marOR loci and acrR, acrS genes were assessed by sequencing. The six strains and their wild-type ramR gene complemented derivatives were analysed by (i) qRT-PCR for gene expression of regulatory and efflux genes and by (ii) MIC determinations of quinolones, FQ and florfenicol as other substrate of AcrAB-TolC. Results. All serotype Kentucky strains studied were of sequence type X1-ST198, excepted the 98K strain which was X4-ST198. Among the thirty S. Kentucky strains, three overproduce AcrAB-TolC (3). All three strains presented different mutations in the ramR gene in comparison to the reference strain 98K. The three detected mutations (deletion of 91 bp, insertion of 1 bp or 4 bp) resulted in frame shift of the ramR gene. All other efflux regulatory genes were not mutated. As confirmed by complementation with a wild-type ramR gene, all three mutations were responsible for increased expression of ramA and acrAB. Increased expression of tolC and acrEF genes was observed in 2 out of the 3 strains. All three mutations were shown to increase two-fold the MICs of FQ and florfenicol in comparison to the ramR complemented derivatives and the reference strain. The two strains with a basal expression level of AcrAB-TolC presented any mutation in sequenced genes. The only strain presenting 3 substitutions in QRDR associated with 1 mutation in ramR upregulating AcrAB-TolC leaded to the higher level FQ resistance Conclusion. Various novel ramR mutations, responsible for increased efflux, were detected in the emerging epidemic serotype Kentucky ST198 clone. As previously reported in other FQ-resistant strains of serotypes Typhimurium or Schwarzengrund, ramR mutations seem to be sporadic (10 % in this study) and contribute only to a little extent to the decreased FQ susceptibility

National audience

Background. Efflux is a mechanism that has been previously reported to increase fluoroquinolone (FQ) resistance levels when it is upregulated in clinical isolates of Salmonella enterica mainly of serotype Typhimurium (1). In this study efflux related genes were investigated in the emerging FQ-resistant epidemic S. enterica serotype Kentucky ST198 clone (2) for the presence of mutations in their regulatory genes, and for their expression levels. Methods. Among a representative panel of thirty serotype Kentucky strains from Egypt or east Africa with decreased FQ susceptibility, three strains overproducing the AcrAB-TolC efflux system were detected and studied (3). Two FQ-resistant strains with substitutions in QRDR and with basal expression level of AcrABTolC and the susceptible reference strain 98K were used as control. Genetic relatedness was determined by XbaI-pulsed field gel electrophoresis and multilocus sequence typing. Presence of mutations in the ramRA, soxRS, marOR loci and acrR, acrS genes were assessed by sequencing. The six strains and their wild-type ramR gene complemented derivatives were analysed by (i) qRT-PCR for gene expression of regulatory and efflux genes and by (ii) MIC determinations of quinolones, FQ and florfenicol as other substrate of AcrAB-TolC. Results. All serotype Kentucky strains studied were of sequence type X1-ST198, excepted the 98K strain which was X4-ST198. Among the thirty S. Kentucky strains, three overproduce AcrAB-TolC (3). All three strains presented different mutations in the ramR gene in comparison to the reference strain 98K. The three detected mutations (deletion of 91 bp, insertion of 1 bp or 4 bp) resulted in frame shift of the ramR gene. All other efflux regulatory genes were not mutated. As confirmed by complementation with a wild-type ramR gene, all three mutations were responsible for increased expression of ramA and acrAB. Increased expression of tolC and acrEF genes was observed in 2 out of the 3 strains. All three mutations were shown to increase two-fold the MICs of FQ and florfenicol in comparison to the ramR complemented derivatives and the reference strain. The two strains with a basal expression level of AcrAB-TolC presented any mutation in sequenced genes. The only strain presenting 3 substitutions in QRDR associated with 1 mutation in ramR upregulating AcrAB-TolC leaded to the higher level FQ resistance [br/] Conclusion. Various novel ramR mutations, responsible for increased efflux, were detected in the emerging epidemic serotype Kentucky ST198 clone. As previously reported in other FQ-resistant strains of serotypes Typhimurium or Schwarzengrund, ramR mutations seem to be sporadic (10 % in this study) and contribute only to a little extent to the decreased FQ susceptibility