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International audience; Objective: The 23 French Regional Pneumococcal Observatories (ORPs) analyzed antibiotic resistance and serotypes of Streptococcus pneumoniae strains isolated from invasive infections in France over a 12-year period.Methods: Between 2009 and 2021, the ORPs analyzed 19,319 strains, including 1,965 in children and 17,354 in adults. Strains were assessed for their resistance to penicillin G, amoxicillin and cefotaxime. Serotypes were identified in collaboration with the National Reference Centre.Results: During this period, the number of strains collected yearly decreased significantly. The decrease was particularly pronounced up until 2013, especially in children (-61.0%). However, penicillin non-susceptible strains (PNSPs) increased in children (24.7% in 2009 vs 45.0% in 2021, p < 0.0001) and in adults (27.1% in 2009 vs 31.3% in 2021, p < 0.05), as well as resistance (I + R) to amoxicillin (children: 12.5% in 2009 vs 19.4% in 2021, p < 0.05; adults: 13.4% in 2009 vs 14.5% in 2021, NS) and resistance (I + R) to cefotaxime (children: 8.0% in 2009 vs 13.1% in 2021, p < 0.05; adults: 7.1% in 2009 vs 11.9% in 2021, p < 0.0001). All in all, the proportion of strains belonging to serotypes present in the PCV13 vaccine has fallen sharply, from 64.8% in 2009 to 23.6 % in 2021. At the same time, serotypes such as 8, 10A, 11A, 15B/C and 9N, not included in PCV13, were increasing.Conclusion: During the study period, data collected by the network highlighted an increase of invasive PNSPs in children and non-vaccine serotypes. Surveillance of resistance and serotypes remains instrumental, particularly to monitor the evolution of vaccine efficacy.

The fosfomycin breakpoint using the disc diffusion method (DDM) changed in the 2019 CA-SFM/EUCAST guidelines v2 (24mm versus 19mm). We assessed its impact on categorization of Enterobacterales recovered from urine samples in emergency departments. A total of 7749 and 2348 strains were tested using the DDM and the broth microdilution method (BMD), respectively. The DDM with the 19-mm breakpoint was in accordance with the BMD. Using the 24-mm breakpoint, the overall rate of fosfomycin resistance in Enterobacterales increased by three-fold (5.6% vs 18.1%, P0.01) and reached 2.8% and 86.5% in E. coli and K. pneumoniae, respectively. French guidelines for the management of community-acquired UTI remain appropriate. The accuracy of the methods for routine fosfomycin susceptibility testing should be assessed. The role of fosfomycin in the treatment of documented CA-UTI due to Enterobacterales other than E. coli should be evaluated considering its rate of resistance and recent data reporting low accuracy.

This study compared standard of care testing (SOC) to BioFire® FilmArray® Pneumonia plus Panel (PNplus). PNplus detects 15 bacteria with semiquantitative log bin values, 7 antibiotic resistance markers, three atypical bacteria (AB), and eight viral classes directly from bronchoalveolar lavage-like specimens (BLS) and sputum-like specimens (SLS). Fifty-two laboratories from 13 European countries and Israel tested 1234 BLS and 1242 SLS with PNplus and SOC. Detection rates and number of pathogens/samples were compared for PNplus pathogens. PNplus bin values and SOC quantities were compared. Three thousand two hundred sixty-two bacteria in PNplus were detected by PNplus and/or SOC. SOC detected 57.1% compared to 95.8% for PNplus (p ≤ 0.0001). PNplus semiquantitative bin values were less than SOC, equal to SOC, or greater than SOC in 5.1%, 25.4%, and 69.6% of results, respectively. PNplus bin values were on average ≥ 1 log than SOC values (58.5% 1–2 logs; 11.0% 3–4 logs). PNplus identified 98.2% of MRSA and SOC 55.6%. SOC detected 73/103 AB (70.9%) and 134/631 viruses (21.2%). PNplus detected 93/103 AB (90.3%) and 618/631 viruses (97.9%) (p ≤ 0.0001). PNplus and SOC mean number of pathogens/samples were 1.99 and 1.44, respectively. All gram-negative resistance markers were detected. PNplus and SOC results were fully or partially concordant for 49.1% and 26.4% of specimens, respectively. PNplus was highly sensitive and detected more potential pneumonia pathogens than SOC. Semiquantification may assist in understanding pathogen significance. As PNplus generates results in approximately 1 h, PNplus has potential to direct antimicrobial therapy in near real time and improve antimicrobial stewardship and patient outcomes.

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.