Molecular and functional analysis of the novel cfr(D) linezolid resistance gene identified in Enterococcus faecium.

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).

[Microbiological testing of flexible endoscopes: contribution of the approach of accreditation in the implementation of regulation]. / Analyse microbiologique des solutions de contrôle d'endoscope : apport de la démarche d'accréditation dans la mise en application de la réglementation.

The microbiological surveillance of flexible endoscopes reprocessing involves counts of the mesophilic total aerobic revivable flora and the detection of indicator microorganisms of dysfunction. We presented the assay at the COFRAC certification, to ensure a maximal level of confidence in the quality of our results. The existing quality system management was used by the type B widened flexible scope for data from previous accreditations for the identification of Staphylococcus aureus, Enterococci and Enterobacteriaceae. The quality insurance of the results and the technical authorization were guaranteed by the participation at a program of interlaboratory comparisons and the elaboration of internal quality controls. Risks analyses and conventions were wrote with endoscopy sectors. The visit led the opening of a single and not critical deviation sheet on the control of consumables and the accreditation for the analysis was pronounced.

[Optimization of incubation duration of culture media in microbiology]. / Optimisation des durées d'incubation des milieux de culture en microbiologie.

In order to perform biological analysis, clinical laboratories apply the instructions of reagent suppliers. For culture media these instructions are often incomplete and poorly adapted to the variety of clinical samples and micro-organisms. The REMIC can help to overcome these shortcomings. Required time of incubation for culture media are proposed based on the nature of the sample and the type of micro-organism suspected. Nevertheless, they are most often expressed in multiple of 24 hours and they are often considered as minimal by the laboratories. As the samples are inoculated "continuously", while the readings are most often done at a single definite time of the day, we propose a strategy to optimize incubation duration of cultures medium. A time of incubation in the day so-called "limit" is defined. From this, the incubations are stopped or prolonged according to the results of the culture and the direct examination. As the instructions of suppliers of culture media are not adapted, it appears necessary that these suppliers relies on the repositories of professional societies as this is the case for agars medias used for antibiotic susceptibility testing.

[Accreditation of the screening for the rectal carriage of vanA and/or vanB Vancomycin-resistant enterococci by an accredited laboratory under the standard NF EN ISO/IEC 17025:2005]. / Accréditation du dépistage du portage rectal des entérocoques résistants aux glycopeptides vanA et/ou vanB par un laboratoire déjà accrédité en portée B selon la norme NF EN ISO/CEI 17025:2005.

The quality of the screening of vanA and/or vanB Vancomycin-resistant enterococcal (VRE) carriage by patients transferred from foreign countries plays a role in the management of risks linked to extensively drug resistant organisms (XDRO). Accreditation of the screening according to the NF EN ISO 15189 and NF EN ISO/IEC 17025 standards contributes to satisfy the level of quality. Our laboratory was already accredited according to the NF EN ISO/IEC 17025 standard. We used its quality management system and the type B widened flexible scope to identify the required criteria based on microbiology and infection control standards and those of Afnor and Cofrac, and to validate the screening procedure. Accreditation was obtained for use of the Type B scope, for culture-based detection and identification (codes BA1 and BA5), for determination of the minimal inhibitory concentrations of glycopeptides (code BA6), and for the detection of resistance genes to glycopeptides by polymerase chain reaction (code BA8). The maturity of our quality management system contributed to validate the screening procedures following the required criteria of the NF EN ISO/IEC 17025 standard.

[Evaluation of the environmental contamination in microbiology Which strategy to adopt?] / Évaluation de la contamination environnementale en microbiologie Quelle stratégie adopter ?

The culture of micro-organisms exposes to the risk of microbiological contamination at all stages of the analysis: inoculation on culture media, incubation, and observation of cultures. During our accreditation renewal audit, a surveillance point was notified, regarding the lack of consideration of the risk of microbiological contamination. Its mastery mainly relies on cleaning/disinfection operations and their traceability. In addition, several strategies based on environmental sampling or indicators can be performed. We propose a risk analysis in order to present these strategies.

Control of legionellae in a new healthcare facility following implementation of a thermal control strategy.

INTRODUCTION: The management of the Legionella risk in hospitals is essentially related to preventive measures of the hot-water supplies. AIM: To monitor the control of legionellae before and after moving to a new hospital facility. METHODS: We implemented a survey program based on the surveillance of the temperature of the hot-water supply and detection and counting of Legionella pneumophila and Legionella spp. by quantitative polymerase chain reaction and culture methods. RESULTS: Our survey program revealed that the hot-water system was colonized by L. pneumophila and Legionella spp. before the arrival of the first patients, despite the implementation of preventive measures. Thus, maintenance on the hot-water production system and subsequent cleaning and superheat disinfection of the hot-water supplies were performed, leading to the eradication of L. pneumophila reservoirs and the decrease of Legionella spp. reservoirs. No reservoirs of L. pneumophila and only rare persistent reservoirs of Legionella spp. were detected after the transfer of hospitalized patients to the new healthcare facility and during the following four years, demonstrating the effectiveness of our corrective measures, without using biocides. L. anisa was identified as the only strain of viable and cultivable Legionella spp. and was undetected during the last year. CONCLUSIONS: The strict application of our survey program before and after moving to the new hospital associated with strict implementation of corrective measures allowed us to efficiently manage the Legionella-linked risk during this period.

Problems associated with the microbiological control of the breast milk from hospital milk bank units.

The microbiological tests on breast milk performed when samples of pasteurized breast milk are added to hospital milk banks are covered by French regulations dating from December 3rd 2007. They involve counts of the aerobic total flora and of Staphylococcus aureus in a sample of milk before pasteurization, and culture after pasteurization to check that the treated milk is sterile. The regulations specify the nature of the agar plates to be used, together with the conditions for plating and incubation, but they lack detail in other areas. We developed a quality assurance system, modified our process to meet the statutory requirements, prepared for COFRAC certification of the laboratory for this parameter, and proposed solutions to overcome the inadequacies of the regulations. The modifications of the process associated with the quality approach led to a successful initial certification visit. However, the preparation for this certification highlighted other inadequacies of the regulations that might affect the final results obtained for total flora and S. aureus counts. We think that the text should be modified to overcome these problems and to ensure high-quality counting such that those running hospital milk banks can have confidence in the laboratory results they receive.

[Accreditation of a hygiene hospital laboratory for sampling and analysis activities for the detection and counting of Legionella in water]. / Accréditation d'un laboratoire d'hygiène hospitalière pour le prélèvement et l'analyse de recherche et dénombrement des légionelles dans l'eau.

Since January 1(st) 2012, detection and counting of Legionella bacteria have been obligatory in France and must be carried out by COFRAC-accredited laboratories. In our establishment, sampling and analysis were outsourced and our hospital was scheduled to move to a new site. We aimed to develop both these activities in-house and to obtain COFRAC accreditation, whilst organizing the move to the new site. We set up a quality assurance system bringing together staff from the hygiene laboratory and institutional resource managers. We set up sampling and analysis activities in-house 13 months before requesting accreditation. The initial evaluation took place before we moved and identified 17 areas of deficiency, six of which were considered critical. After we had moved, a subsequent evaluation considered 14 of these deficiencies to have been corrected, included the six initially identified as critical. We were therefore awarded accreditation. The quality assurance system established during the year before our request was submitted led to accreditation two and a half years after the transfer in-house of sampling and analysis activities, despite our hospital moving during this period.

Human cytomegalovirus infection reduces surface CCR5 expression in human microglial cells, astrocytes and monocyte-derived macrophages.

Within the brain, glial cells are target cells for human cytomegalovirus (HCMV) and HIV. We infected cultures of unstimulated human microglial cells and astrocytes of embryonic origin and of monocyte-derived macrophages (MDM) with HCMV strain AD169 and observed down-regulation of the plasma membrane expression of CCR5 in the three cell types, and of CXCR4 and CD4 in microglial cells only. Cells were then coinfected simultaneously or at a 24-h interval with both AD169 and two different HIV-1 monocytotropic strains. HCMV late antigens and HIV-1 tat protein colocalized in the cytoplasm of 5-10% of microglia and MDM. p24 antigen levels decreased 10- to 40-fold in supernatants of MDM and the reduction was greater when HCMV infection was performed 24 h before HIV-1 infection. These data suggest that HCMV-induced reduction in the cell-surface expression of the primary co-receptor of HIV-1 monocytotropic strains may impair the ability of HIV to infect these cells.