Meropenem heteroresistance in clinical isolates of OXA-48-producing Klebsiella pneumoniae.

OXA-48-producing Klebsiellapneumoniae isolates often show growth of colonies within inhibition zones in carbapenem diffusion assays. The nature of these colonies was investigated in a series of clinical isolates of OXA-48-producing K. pneumoniae obtained in the context of a hospital outbreak, and they were found to be persistent colonies that reproduced again the same phenotype when they were collected and tested in diffusion assays again. The frequency of mutations conferring resistance to meropenem (8 µg/mL) was determined for the same isolates. The average mutation frequency was 5.47·10-6 (range: 2.59·10-8-5.87·10-5), and the analysis of several resistant mutants showed that all of them had mutations in the ompK36 porin gene. Heteroresistance was investigated using population analysis profiling. The profiles were compatible with mutation frequency assays, and all the colonies analyzed were resistant mutants. In OXA-48-producing K. pneumoniae, the growth of persisters seems to be specific of diffusion assays.

Genomic path to pandrug resistance in a clinical isolate of Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae have spread globally throughout tertiary hospitals. Many Carbapenem-resistant K. pneumoniae clinical isolates are multidrug-resistant (MDR) and may become eventually pandrug-resistant (PDR). Here we present the closed genome of a PDR VIM-1-producing K. pneumoniae strain (KP1050) obtained in a tertiary hospital. The isolate belonged to sequence type 54 (ST54) and had five extrachromosomal elements (four plasmids and a circular phage genome). Most of the antimicrobial resistance genes (ARGs) were located in two clusters borne by two of the plasmids, comprising a class 1 integron that contained up to 14 ARGs including a VIM-1 metallo-ß-lactamase gene, and an IS26 transposon that contained a mobile element from Acinetobacter baumannii encoding the amikacin resistance gene aac(6')-Ian. A MDR isolate obtained 6 years previously was identified (KP1048) retrospectively and was sequenced. Comparison of the two genomes showed that chromosomal mutations in outer membrane porins as well as mutations in the ramR and phoQ genes contributed to increase the resistance spectrum.

Analysis of drug resistance mutations in whole blood DNA from HIV-1 infected patients by single genome and ultradeep sequencing analysis.

In HIV-1 infected patients blood CD4+ T lymphocytes could be a valuable target to analyse drug resistance mutations (DRM) selected over the course of the infection. However, detection of viral resistance mutations in cellular DNA by standard genotype resistance techniques (SGRT) is suboptimal. Whole blood DNA (wbDNA) from 12 HIV-1 infected patients on ART was studied by Single Genome Sequencing (SGS) and 8 of them also by Ultradeep pyrosequencing (UDP). Results were compared with contemporary and historical DRM detected in plasma by SGRT during follow up. All the contemporary DRM detected in plasma from the viremic patients were detected by SGS and UDP (20 from 7 patients and 4 from 5 patients respectively). Out of the 67 historical DRM detected in plasma and no longer present at the time of testing, 38 (57%) were detected by SGS in 12 patients and 27 out of 46 (59%) by UDP in 8 patients. Additional DRM never reported in plasma by SGRT were detected by SGS (12 from 8 patients) and UDP (10 from 8 patients). Analysis of wbDNA from HIV-1 infected patients by SGS and UDP provides proof of concept of the value of blood DNA to investigate current and archived DRM in HIV-1 infected patients on ART.

Development of daptomycin resistance during therapy in a patient with methicillin-resistant Staphylococcus aureus endocarditis: a case report / Desarrollo de resistencia a daptomicina durante el tratamiento de un paciente con endocarditis por Staphylococcus aureus resistente a meticilina

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Bacteriemia por Nocardia veterana tras una infección pulmonar en un paciente con leucemia mieloide aguda / Nocardia veterana bacteraemia after a lung infection in a patient with acute myeloid leukaemia

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Label-free DNA-based detection of Mycobacterium tuberculosis and rifampicin resistance through hydration induced stress in microcantilevers.

We have developed a label-free assay for the genomic detection of Mycobacterium tuberculosis and rifampicin resistance. The method relies on the quantification of the hydration induced stress on microcantilever biosensors functionalized with oligonucleotide probes, before and after hybridization with specific targets. We have found a limit of detection of 10 fg/mL for PCR amplified products of 122 bp. Furthermore, the technique can successfully target genomic DNA (gDNA) fragments of length >500 bp, and it can successfully discriminate single mismatches. We have used both loci IS6110 and rpoB as targets to detect the mycobacteria and the rifampicin resistance from gDNA directly extracted from bacterial culture and without PCR amplification. We have been able to detect 2 pg/mL target concentration in samples with an excess of interfering DNA and in a total analysis time of 1 h and 30 min. The detection limit found demonstrates the capability to develop direct assays without the need for long culture steps or PCR amplification. The methodology can be easily translated to different microbial targets, and it is suitable for further development of miniaturized devices and multiplexed detection.

Clinical evaluation of a disposable amperometric magneto-genosensor for the detection and identification of Streptococcus pneumoniae.

A disposable PCR-based amperometric magneto-genosensor for detection and identification of Streptococcus pneumoniae was evaluated. ROC curve analysis used to determine optimal signal cutoff values yielded a sensitivity of 91% and a specificity of 90%. The method was also tested for the direct detection of pneumococci in clinical samples.