Absorption/Attenuation Spectral Description of ESKAPEE Bacteria: Application to Seeder-Free Culture Monitoring, Mammalian T-Cell and Bacteria Mixture Analysis and Contamination Description.

Despite numerous innovations, measuring bacteria concentrations on a routine basis is still time consuming and ensuring accurate measurements requires careful handling. Furthermore, it often requires sampling small volumes of bacteria suspensions which might be poorly representative of the real bacteria concentration. In this paper, we propose a spectroscopy measurement method based on a description of the absorption/attenuation spectra of ESKAPEE bacteria. Concentrations were measured with accuracies less than 2%. In addition, mixing the mathematical description of the absorption/attenuation spectra of mammalian T-cells and bacteria allows for the simultaneous measurements of both species' concentrations. This method allows real-time, sampling-free and seeder-free measurement and can be easily integrated into a closed-system environment.

Absorption Spectra Description for T-Cell Concentrations Determination and Simultaneous Measurements of Species during Co-Cultures.

Advanced Therapy Medicinal Products are promising drugs for patients in therapeutic impasses. Their complex fabrication process implies regular quality controls to monitor cell concentration. Among the different methods available, optical techniques offer several advantages. Our study aims to measure cell concentration in real time in a potential closed-loop environment using white light spectroscopy and to test the possibility of simultaneously measuring concentrations of several species. By analyzing the shapes of the absorption spectra, this system allowed the quantification of T-cells with an accuracy of about 3% during 30 h of cultivation monitoring and 26 h of doubling time, coherent with what is expected for normal cell culture. Moreover, our system permitted concentration measurements for two species in reconstructed co-cultures of T-cells and Candida albicans yeasts. This method can now be applied to any single or co-culture, it allows real-time monitoring, and can be easily integrated into a closed system.

Molecular characterization of blaNDM-1 in a sequence type 235 Pseudomonas aeruginosa isolate from France.

An NDM-1 carbapenemase-producing Pseudomonas aeruginosa isolate was recovered from a patient hospitalized in France after a previous hospitalization in Serbia. Genetic studies revealed that the blaNDM-1 gene was surrounded by insertion sequence ISAba125 and a truncated bleomycin resistance gene. This blaNDM-1 region was a part of the variable region of a new complex class 1 integron bearing IS common region 1 (ISCR1). The presence of ISPa7 upstream of this integron suggests insertion in a chromosomally located Tn402-like structure.

Complexity of resistance mechanisms to imipenem in intensive care unit strains of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa can become resistant to carbapenems by both intrinsic (mutation-driven) and transferable (ß-lactamase-based) mechanisms. Knowledge of the prevalence of these various mechanisms is important in intensive care units (ICUs) in order to define optimal prevention and therapeutic strategies. METHODS: A total of 109 imipenem-non-susceptible (MIC >4 mg/L) strains of P. aeruginosa were collected in June 2010 from the ICUs of 26 French public hospitals. Their resistance mechanisms were characterized by phenotypic, enzymatic, western blotting and molecular methods. RESULTS: Single or associated imipenem resistance mechanisms were identified among the 109 strains. Seven isolates (6.4%) were found to produce a metallo-ß-lactamase (one VIM-1, four VIM-2, one VIM-4 and one IMP-29). Porin OprD was lost in 94 (86.2%) strains as a result of mutations or gene disruption by various insertion sequences (ISPa1635, ISPa1328, IS911, ISPs1, IS51, IS222 and ISPa41). Thirteen other strains were shown to be regulatory mutants in which down-regulation of oprD was coupled with overexpressed efflux pumps CzcCBA (n = 1), MexXY (n = 9) and MexEF-OprN (n = 3). The lack of OprD was due to disruption of the oprD promoter by ISPsy2 in one strain and alteration of the porin signal sequence in another. CONCLUSIONS: Imipenem resistance in ICU P. aeruginosa strains may result from multiple mechanisms involving metallo-ß-lactamase gene acquisition and genetic events (mutations and ISs) inactivating oprD, turning down its expression while increasing efflux activities or preventing insertion of porin OprD in the outer membrane. This diversity of mechanisms allows P. aeruginosa, more than any other nosocomial pathogen, to rapidly adapt to carbapenems in ICUs.

IMP-29, a novel IMP-type metallo-ß-lactamase in Pseudomonas aeruginosa.

Analysis of two clonally related multiresistant Pseudomonas aeruginosa isolates led to the identification of a novel IMP-type metallo-ß-lactamase. IMP-29 was significantly different from the other IMP variants (the closest variant being IMP-5 with 93% amino acid identity). The bla(IMP-29) gene cassette was carried by a class 1 integron in strain 10.298, while in strain 10.266 it was located in a rearranged DNA region on a 30-kb conjugative plasmid. Biochemical analysis confirmed that IMP-29 efficiently hydrolyzed carbapenems.