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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.