Sensibilité aux antibiotiques des biofilms de souches de pseudomonas aeruginosa isolées de patients atteints de mucoviscidose

NICE-BU Médecine Odontologie (060882102) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

New lnu(C) Gene Conferring Resistance to Lincomycin by Nucleotidylation in Streptococcus agalactiae UCN36

Streptococcus agalactiae UCN36 was resistant to lincomycin (MIC = 16 μg/ml) but susceptible to clindamycin (MIC = 0.12 μg/ml) and erythromycin (MIC = 0.06 μg/ml). A 4-kb HindIII fragment was cloned from S. agalactiae UCN36 total DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to lincomycin. The sequence analysis of the fragment showed the presence of a 1,724-bp element delineated by imperfect inverted repeats (22 of 25 bp) and inserted in the operon for capsular synthesis of S. agalactiae UCN36. This element carried two open reading frames (ORF). The deduced amino acid sequence of the upstream ORF displayed similarity with transposases from anaerobes and IS1. The downstream ORF, lnu(C), encoded a 164-amino-acid protein with 26% to 27% identity with the LnuA(N2), LnuA, and LnuA′ lincosamide nucleotidyltransferases reported for Bacteroides and Staphylococcus, respectively. Crude lysates of E. coli AG100A containing the cloned lnu(C) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl(2). Mass spectrometry experiments demonstrated that the LnuC enzyme catalyzed adenylylation of lincomycin

Emergence of Macrolide Resistance Gene mph(B) in Streptococcus uberis and Cooperative Effects with rdmC-Like Gene▿

Streptococcus uberis UCN60 was resistant to spiramycin (MIC = 8 μg/ml) but susceptible to erythromycin (MIC = 0.06 μg/ml), azithromycin (MIC = 0.12 μg/ml), josamycin (MIC = 0.25 μg/ml), and tylosin (MIC = 0.5 μg/ml). A 2.5-kb HindIII fragment was cloned from S. uberis UCN60 DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to spiramycin by inactivation. The sequence analysis of the fragment showed the presence of an rdmC-like gene that putatively encoded a protein belonging to the alpha/beta hydrolase family and of the first 196 nucleotides of the mph(B) gene putatively encoding a phosphotransferase known to inactivate 14-, 15-, and 16-membered macrolides in E. coli. The entire mph(B) gene was then identified in S. uberis UCN60. The two genes were expressed alone or in combination in E. coli, Staphylococcus aureus, and Enterococcus faecalis. Analysis of MICs revealed that rdmC-like alone did not confer resistance to erythromycin, tylosin, and josamycin in those three hosts. It conferred resistance to spiramycin in E. coli and E. faecalis but not in S. aureus. mph(B) conferred resistance in E. coli to erythromycin, tylosin, josamycin, and spiramycin but only low levels of resistance in E. faecalis and S. aureus to spiramycin (MIC = 8 μg/ml). The combination of mph(B) and rdmC-like genes resulted in a resistance to spiramycin and tylosin in the three hosts that significantly exceeded the mere addition of the resistance levels conferred by each resistance mechanism alone

Lincomycin Resistance Gene lnu(D) in Streptococcus uberis▿

Streptococcus uberis UCN 42, isolated from a case of bovine mastitis, was intermediately resistant to lincomycin (MIC = 2 μg/ml) while remaining susceptible to clindamycin (MIC = 0.06 μg/ml) and erythromycin. A 1.1-kb SacI fragment was cloned from S. uberis UCN 42 total DNA on plasmid pUC 18 and introduced into Escherichia coli AG100A, where it conferred resistance to both clindamycin and lincomycin. The sequence analysis of the fragment showed the presence of a new gene, named lnu(D), that encoded a 164-amino-acid protein with 53% identity with Lnu(C) previously reported to occur in Streptococcus agalactiae. Crude lysates of E. coli AG100A containing the cloned lnu(D) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl2. Mass spectrometry experiments demonstrated that the lnu(D) enzyme catalyzed adenylylation of clindamycin. A domain conserved in deduced sequences of lincosamide O-nucleotidyltransferases Lnu(A), Lnu(C), LinAN2, and Lin(D) and in the aminoglycoside nucleotidyltransferase ANT(2′′) was identified

Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a couple of probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction steps. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the Biomark instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring in addition to SARS-CoV-2 probes of other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). Its 10 nL range volume is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several procedures, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities

Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

International audienceThe emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction vo