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Background Despite the fact that carbapenem-resistant Enterobacterales (CRE) mostly cause urinary tract infections (UTIs), only few studies have focused on the efficacity of mecillinam against these CRE. Objectives To evaluate the mecillinam susceptibility of a huge collection of CRE, including carbapenemase-producing Enterobacterales (CPE) and non-CPE (ESBL and AmpC producers with decreased permeability of the outer membrane). Methods A total of 8310 non-duplicate clinical CRE, including 4042 OXA-48-like producers, 1094 NDM producers, 411 VIM producers, 174 KPC producers, 42 IMI producers, 153 multiple-carbapenemase producers and 45 isolates producing other types of carbapenemases (such as IMP-like enzymes or GES-5), were included in the study. WGS was performed on all CPE using Illumina technology. Categorization of susceptibility to mecillinam was performed using disc diffusion (mecillinam discs at 10 μg; I2A, France) according to EUCAST recommendations. The results were interpreted according to EUCAST guidelines (S ≥15 mm). Results Significantly higher susceptibility rates were observed for carbapenem-resistant Proteus spp. (85%) and carbapenem-resistant Escherichia coli (84%), which are the two most common species responsible for UTIs, than for Klebsiella pneumoniae (67%), Enterobacter cloacae complex (75%), Citrobacter spp. (65%), Serratia spp. (34%) and Morganella morganii (12%). Susceptibility rates were 84%, 71% and 91% for OXA-48-like, NDM and IMI producers and 70% for non-CPE CRE. Mecillinam was less active against VIM and KPC producers (14% and 0%, respectively). Conclusions Mecillinam might be an alternative for the treatment of infections due to CRE, particularly UTIs, except for VIM and KPC producers and for M. morganii and Serratia spp species.

Background: KPC-producing Klebsiella pneumoniae of clonal group 258 are prominent in healthcare settings in many regions of the world. The blaKPC gene is mostly carried by a multireplicon IncFIIk-IncFI plasmid suspected to be highly compatible and stable in this genetic background. Here, we analysed the genetic diversity of an ST512 K. pneumoniae population in a single patient. Methods: Twelve K. pneumoniae isolates (n = 5 from urine samples and n = 7 from rectal swabs) were recovered from one patient over a 2 month period. Antimicrobial susceptibility testing, plasmid extraction and WGS were performed on all isolates. The first K. pneumoniae isolate, D1, was used as a reference for phylogenetic analysis. Results: Antimicrobial susceptibility testing, plasmid analysis and WGS revealed concomitant carriage of carbapenem-resistant and carbapenem-susceptible K. pneumoniae isolates of ST512, with the absence of the entire blaKPC-carrying plasmid in the susceptible population. Furthermore, 14 other genetic events occurred with- in the genome, including 3 chromosomal deletions (of 71 kb, 33 kb and 11 bp), 2 different insertions of ISKpn26 and 9 SNPs. Interestingly, most of the events occurred in the same chromosomal region that has been deleted independently several times, probably after homologous recombination involving 259 bp repeated sequences. Conclusions: Our study revealed (to the best of our knowledge) the first case of in vivo blaKPC-carrying plasmid curing and a wide within-patient genetic diversity of a single K. pneumoniae ST512 clone over a short period of carriage. This within-patient diversity must be taken into account when characterizing transmission chains using WGS during nosocomial outbreaks., This work was partially funded by the University Paris-Sud, France. L.D., T.N. and R.A.B. are members of the Laboratory of Excellence in Research on Medication and Innovative Therapeutics (LERMIT) supported by a grant from the French National Research Agency (ANR-10-LABX-33), by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) DesInMBL (ANR-14-JAMR-002) and by the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement No. 773830 (Project MedVetKlebs, One Health EJP).

Background OXA-244, a single amino acid variant of OXA-48, demonstrates weaker hydrolytic activity towards carbapenems and temocillin compared with OXA-48. Of note, these antimicrobials are present in high concentrations in several carbapenemase-producing Enterobacterales (CPE) screening media. As a result, some screening media fail to grow OXA-244-producing isolates, while the prevalence of OXA-244 producers is constantly increasing in France. Methods Here, we evaluate the performance of three commercially available CPE screening media [ChromID® CARBA SMART (bioMérieux), Brilliance™ CRE (Thermo Fisher) and mSuperCARBA™ (MAST Diagnostic)] for their ability to detect OXA-244 producers (n = 101). As OXA-244 producers may also express an ESBL, two additional ESBL screening media were tested (Brilliance™ ESBL and ChromID® BLSE). MICs of temocillin and imipenem were determined by broth microdilution. The clonality of OXA-244-producing Escherichia coli isolates (n = 97) was assessed by MLST. Results Overall, the sensitivity of the ChromID® CARBA SMART, Brilliance™ CRE and mSuperCARBA™ media were 14% (95% CI = 8.1%–22.5%), 54% (95% CI = 43.3%–63.4%) and 99% (95% CI = 93.8%–100%), respectively, for the detection of OXA-244 producers. Among the 101 OXA-244-producing isolates, 96% were E. coli and 77%–78% grew on ESBL screening media. MLST analysis identified five main STs among OXA-244-producing E. coli isolates: ST38 (n = 37), ST361 (n = 17), ST69 (n = 12), ST167 (n = 11) and ST10 (n = 8). Conclusions Our results demonstrated that the mSuperCARBA™ medium is very efficient in the detection of OXA-244 producers, unlike the ChromID® CARBA SMART medium. The high prevalence of ESBLs among OXA-244 producers allowed detection of 77%–78% of them using ESBL-specific screening media.

International audience; Objectives: Patient-and procedure-related changes in modern medicine have turned CoNS into one of the major nosocomial pathogens. Treatments of CoNS infections are challenging owing to the large proportion of MDR strains and oxazolidinones often remain the last active antimicrobial molecules. Here, we have investigated a long-lasting outbreak (2010-13) due to methicillin-and linezolid-resistant (LR) CoNS (n " 168), involving 72 carriers and 49 infected patients. Methods: Antimicrobial susceptibilities were tested by the disc diffusion method and MICs were determined by broth microdilution or Etest. The clonal relationship of LR Staphylococcus epidermidis (LRSE) was first determined using a semi-automated repetitive element palindromic PCR (rep-PCR) method. Then, WGS was performed on all cfr-positive LRSE (n " 30) and LRSE isolates representative of each rep-PCR-defined clone (n " 17). Self-transferability of cfr-carrying plasmids was analysed by filter-mating experiments. Results: This outbreak was caused by the dissemination of three clones (ST2, ST5 and ST22) of LRSE. In these clones, linezolid resistance was caused by (i) mutations in the chromosome-located genes encoding the 23S RNA and L3 and L4 ribosomal proteins, but also by (ii) the dissemination of two different self-conjugative plasmids carrying the cfr gene encoding a 23S RNA methylase. By monitoring linezolid prescriptions in two neighbouring hospitals, we highlighted that the spread of LR-CoNS was strongly associated with linezolid use. Conclusions: Physicians should be aware that plasmid-encoded linezolid resistance has started to disseminate among CoNS and that rational use of oxazolidinones is critical to preserve these molecules as efficient treatment options for MDR Gram-positive pathogens.

Objectives KPC-producing pathogens exhibit variable carbapenem susceptibility levels, which is probably the result of the genetic environment of the bla KPC genes. Here we determined the transcriptional start sites (TSSs) and the expression of the bla KPC-2 gene in various genetic contexts and in different hosts ( Escherichia coli , Pseudomonas aeruginosa and Acinetobacter baumannii ). Methods The bla KPC-2 genes along with the upstream sequences derived from Tn 4401b (structure A), Tn 4401b interrupted by Tn 3 /IS 26 (structure B) and Tn 4401b interrupted by Tn 5563 (structure C) were cloned in two E. coli shuttle vectors (pBBR1MCS.3 for expression studies in P. aeruginosa and pIM-arr2 for expression studies in A. baumannii ). MICs were determined by Etests. 5' RACE (where RACE stands for rapid amplification of cDNA ends) and quantitative RT-PCR experiments were performed to determine TSSs and transcription levels, respectively. Results Depending on the bacterial host, different promoters were used for bla KPC-2 gene expression. The highest transcriptional level was obtained in P. aeruginosa with structure C, described only in P. aeruginosa . Tn 4401b (structure A), harbouring two promoters (P1 and P2), was the most efficient in E. coli and A. baumannii . This structure was also efficient in P. aeruginosa , although the same deduced promoter was not used (P1, instead of P2 used by E. coli and A. baumannii ). Two novel TSSs and putative promoters (P2b and P3b) were identified in structure B. In this structure, P2b and P3b were preferably used in E. coli and in P. aeruginosa , respectively, whereas P1 was used in A. baumannii . Conclusions We determined the preferred TSSs of the bla KPC gene in each species and described two novel deduced promoters in structure B.

Objectives: In silico analysis identified a metallo-b-lactamase (MBL) in Erythrobacter litoralis HTCC2594, sharing 55% amino acid identity with NDM-1. The aim of this work was to characterize the chromosomally encoded MBLs from several Erythrobacter spp. that may represent potential reservoirs of acquired MBLs. Methods: Erythrobacter citreus, Erythrobacter flavus, Erythrobacter longus, Erythrobacter aquimaris and Erythrobacter vulgaris were from the Pasteur Institute collection, France. DNA was extracted and used for shotgun cloning, and b-lactamases were expressed in Escherichia coli. MICs for resulting E. coli recombinant strains were determined by Etest. The deduced amino acid sequences were analysed and compared with BLASTP. Enzymatic activity of bacterial extracts from recombinant E. coli strains was determined by UV spectrophotometry with imipenem (100 mM) as substrate. Results: Resulting E. coli recombinant strains harboured hypothetical MBL-encoding genes. MICs of b-lactams showed decreased susceptibility to carbapenems only for E. coli (pFLA-1) and E. coli (pLON-1), expressing the MBL from E. flavus and E. longus, respectively. MBLs from different Erythrobacter spp. shared weak amino acid identity, ranging from 45% to 75% identity. They differed greatly from that of E. litoralis HTCC2594 (and NDM-1), sharing only 11% – 23% identity. Enzymatic activity against imipenem was detectable but weak in all these recombinant E. coli strains, except E. coli (pFLA-1), in which specific activity was significantly higher. Conclusions: Several chromosomally located MBLs have been identified from Erythrobacter spp. They share weak amino acid identity and are very weakly related to other acquired MBLs (10%–23%).

Objectives To characterize bla(CTX-M-15)-carrying plasmids and lineages of nine strains of Escherichia coli from cattle. Methods Plasmid DNA was analysed using PCR-based replicon typing and plasmid sub-typing schemes, restriction fragment length polymorphism, S1 nuclease-PFGE and Southern hybridization. Strains were characterized by PFGE, multilocus sequence typing, phylogenetic grouping and B2-O25b:H4-ST131 (where ST stands for sequence type) clone screening. Susceptibilities to antimicrobials were determined by agar diffusion and resistance genes were characterized by PCR and sequencing. Results The bla(CTX-M-15) gene was found on F31:A4:B1/IncFII and F2:A-:B-/IncFII plasmids, which have been reported abundantly in humans. On F31:A4:B1/IncFII plasmids, the bla(CTX-M-15) gene was associated with the bla(TEM-1), bla(OXA-1) and aac(6')-Ib-cr resistance genes. The bla(CTX-M-15) gene was also found on IncI1 plasmids of the CC31 clonal complex, recently identified in the human epidemic and virulent E. coli clone O104:H4. None of the cattle isolates belonged to the human and widespread clone B2-O25b:H4/ST131, but were mostly of new STs and of the phylogenetic groups A (n=4), B1 (n=3) or D (n=2). The E. coli isolates harbouring the bla(CTX-M-15)-carrying plasmids were genetically diverse, and were recovered from different geographical locations and farms and at different times. Conclusions This study demonstrates that bla(CTX-M-15)-carrying plasmids from cattle-derived non-ST131 E. coli isolates were highly similar to those found in ST131 E. coli isolates commonly reported in humans. It also exemplifies the key role of plasmids versus clonal dissemination in the spread of the bla(CTX-M-15) gene among cattle, and possibly between E. coli isolates detected in humans and cattle.

In February 1998, 195 patients in the geriatric department of a French hospital were screened for the presence of co-amoxiclav-resistant Klebsiella pneumoniae. Eleven co-amoxiclav-resistant isolates obtained all produced an identical IRT-2 beta-lactamase. These K. pneumoniae isolates were clonally related and harboured a c. 55 kb non-conjugative plasmid encoding a non-class-1 integron-located blaIRT-2 gene. This study underlines that geriatric departments may be a reservoir for antibiotic-resistant strains and that IRT beta-lactamase-producing strains may be nosocomial pathogens.

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