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Objectives To characterize the novel cfr(D) gene identified in an Enterococcus faecium clinical isolate (15-307.1) collected from France. Methods The genome of 15-307.1 was entirely sequenced using a hybrid approach combining short-read (MiSeq, Illumina) and long-read (GridION, Oxford Nanopore Technologies) technologies in order to analyse in detail the genetic support and environment of cfr(D). Transfer of linezolid resistance from 15-307.1 to E. faecium BM4107 was attempted by filter-mating experiments. The recombinant plasmid pAT29Ωcfr(D), containing cfr(D) and its own promoter, was transferred to E. faecium HM1070, Enterococcus faecalis JH2-2 and Escherichia coli AG100A. Results As previously reported, 15-307.1 belonged to ST17 and was phenotypically resistant to linezolid (MIC, 16 mg/L), vancomycin and teicoplanin. A hybrid sequencing approach confirmed the presence of several resistance genes including vanA, optrA and cfr(D). Located on a 103 kb plasmid, cfr(D) encoded a 357 amino acid protein, which shared 64%, 64%, 48% and 51% amino acid identity with Cfr, Cfr(B), Cfr(C) and Cfr(E), respectively. Both optrA and cfr(D) were successfully co-transferred to E. faecium BM4107. When expressed in E. faecium HM1070 and E. faecalis JH2-2, pAT29Ωcfr(D) did not confer any resistance, whereas it was responsible for an expected PhLOPSA resistance phenotype in E. coli AG100A. Analysis of the genetic environment of cfr(D) showed multiple IS1216 elements, putatively involved in its mobilization. Conclusions Cfr(D) is a novel member of the family of 23S rRNA methyltransferases. While only conferring a PhLOPSA resistance phenotype when expressed in E. coli, enterococci could constitute an unknown reservoir of cfr(D).

Despite the introduction of routine vaccination against pertussis for more than a half century, leading to a drastic decline in the number of reported cases, pertussis continues to be an important respiratory disease afflicting unvaccinated infants and previously vaccinated children as well as adults in whom immunity has waned. The diagnosis of pertussis is challenging and accurate laboratory identification of Bordetella infections remains problematic. Common laboratory diagnostic methods used for pertussis diagnosis include culture, direct-fluorescent-antibody testing (DFA), serology and polymerase chain reaction (PCR). Culture of Bordetella pertussis is highly specific but fastidious and has limited sensitivity. DFA provides a much more rapid result, but has the disadvantage of poor sensitivity and specificity. Serology is not useful in infants. In older persons, it is hampered by the limitations of paired sera and it provides mainly a retrospective diagnosis. Such limitations of conventional diagnosis testing have led to the development of PCR assays. Notwithstanding its lack of standardization, PCR has been found to be more sensitive and more specific than other methods. In this report, we aimed to review current knowledge about the available diagnostic methods and tests that accurately diagnose pertussis.

Studying pertussis-like respiratory infections, we report the cases of three infants with evidence of both Bordetella pertussis and Mycoplasma pneumoniae. Bordetella infection was identified by the real-time polymerase chain reaction (RT-PCR) of nasopharyngeal specimens. Neither B. pertussis nor B. parapertussis were recovered on the culture of nasopharyngeal aspirates (NPAs) from any subjects. M. pneumoniae etiology was diagnosed by culture and RT-PCR. The evolution was fatal for all of the subjects. We conclude that, among patients with Bordetella infection, co-infection with another respiratory pathogen is often probable, and these mixed infections might cause a more severe form of illness, sometimes leading to death.

Objectives To describe the prevalence of poxtA among clinical linezolid-resistant enterococci (LRE) collected in France from 2016 to 2020 and to extensively characterize its genetic supports and environments. Methods All LRE clinical isolates received at the National Reference Centre for Enterococci from French hospitals between 2016 and 2020 were included. LRE isolates were screened for linezolid resistance genes (cfr-like, optrA and poxtA) by real-time PCR and phenotypically characterized. A collection of 11 representative poxtA-positive isolates (10 Enterococcus faecium and 1 Enterococcus faecalis) underwent WGS by hybrid assembly combining short-read (Illumina MiSeq) and long-read (MinION) approaches. Transferability of poxtA was attempted by filter-mating experiments. Results Out of 466 LRE received at the National Reference Centre for Enterococci over the period, 47 (10.1%) were poxtA-positive, including 42 E. faecium. The 11 isolates characterized by WGS were confirmed to be epidemiologically unrelated by core genome analysis and eight different STs were assigned to E. faecium isolates. The poxtA gene was found to be plasmid carried and flanked by IS1216E transposase genes in all isolates and frequently linked with fexB, tet(M) and tet(L). A total of seven distinct poxtA-harbouring plasmids were obtained after hybrid assembly and plasmid transfer of poxtA was successful in three cases. For the two poxtA/optrA-positive isolates, those genes were carried by different plasmids. Conclusions The poxtA gene has been circulating among clinical enterococci in France since at least 2016, mostly in E. faecium and independently from optrA. The poxtA-carrying plasmids often co-carried resistance genes to phenicols and tetracyclines, and could have been co-selected through their veterinary use.

Background Linezolid-resistant enterococci (LRE) causing infections that are challenging to treat are rising, highlighting the need for reliable screening of LRE clinical isolates. Objectives To evaluate the ability of the broth microdilution (BMD) method for LRE detection and to assess the performance of seven commercially available techniques for linezolid susceptibility testing. Methods A collection of 100 clinical isolates (80 Enterococcus faecium and 20 Enterococcus faecalis), including 20 optrA-positive isolates, 17 poxtA-positive isolates and 1 optrA/poxtA-positive E. faecium isolate, were studied. MICs were determined after 18 h [Day 1 (D1)] and 42 h [Day 2 (D2)] of incubation and interpreted following EUCAST and CLSI guidelines, which currently provide different interpretative breakpoints. Performance of commercial techniques was compared with BMD results. Results MIC50/D1 and MIC50/D2 were both 8 mg/L, while MIC90/D1 and MIC90/D2 were 16 and 32 mg/L, respectively. MICD1 values for poxtA-positive isolates were lower than those for optrA-positive isolates. Proportions of susceptible isolates at D1 and D2 were 48% and 41%, respectively, according to EUCAST breakpoints and 35% and 13%, respectively, according to CLSI criteria (the proportions of isolates categorized as intermediate following CLSI recommendations were 13% and 28% at D1 and D2, respectively). Percentage susceptibility assessed by the commercially available techniques was always higher. The four commercial methods allowing MIC determination provided an overall essential agreement of ≥90% at D1. Categorical agreement and error rates were generally improved at D2. Conclusions Non-automated methods (Sensititre and UMIC) and, to a lesser extent, gradient strip Etest appear to show an acceptable correlation with the BMD reference method for the detection of isolates with low MICs of linezolid after prolonged incubation.

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