Intensive care antibiotic consumption and resistance patterns: a cross-correlation analysis

Background

Over recent decades, a dramatic increase in infections caused by multidrug-resistant pathogens has been observed worldwide. The aim of the present study was to investigate the relationship between local resistance bacterial patterns and antibiotic consumption in an intensive care unit in a Romanian university hospital.

Methods

A prospective study was conducted between 1st January 2012 and 31st December 2013. Data covering the consumption of antibacterial drugs and the incidence density for the main resistance phenotypes was collected on a monthly basis, and this data was aggregated quarterly. The relationship between the antibiotic consumption and resistance was investigated using cross-correlation, and four regression models were constructed, using the SPSS version 20.0 (IBM, Chicago, IL) and the R version 3.2.3 packages.

Results

During the period studied, the incidence of combined-resistant and carbapenem-resistant P. aeruginosa strains increased significantly [(gradient = 0.78, R² = 0.707, p = 0.009) (gradient = 0.74, R² = 0.666, p = 0.013) respectively], mirroring the increase in consumption of β-lactam antibiotics with β-lactamase inhibitors (piperacillin/tazobactam) and carbapenems (meropenem) [(gradient = 10.91, R² = 0.698, p = 0.010) and (gradient = 14.63, R² = 0.753, p = 0.005) respectively]. The highest cross-correlation coefficients for zero time lags were found between combined-resistant vs. penicillins consumption and carbapenem-resistant P. aeruginosa strains vs. carbapenems consumption (0.876 and 0.928, respectively). The best model describing the relation between combined-resistant P. aeruginosa strains and penicillins consumption during a given quarter incorporates both the consumption and the incidence of combined-resistant strains in the hospital department during the previous quarter (multiple R² = 0.953, p = 0.017). The best model for explaining the carbapenem resistance of P. aeruginosa strains based on meropenem consumption during a given quarter proved to be the adjusted model which takes into consideration both previous consumption and incidence density of strains during the previous quarter (Multiple R² = 0.921, p = 0.037).

Conclusions

The cross-correlation coefficients and the fitted regression models provide additional evidence that resistance during the a given quarter depends not only on the consumption of antibacterial chemotherapeutic drugs in both that quarter and the previous one, but also on the incidence of resistant strains circulating during the previous quarter.

     

Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway

Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.     

Isolation and phenotypic characterisation of stem cells from late stage osteoarthritic mesenchymal tissues

Introduction: Osteoarthritis (OA) represents an increasing health issue worldwide. Regenerative medicine (RM) has raised the hope for introducing revolutionary therapies in clinical practice. Detection of autologus cell sources can improve accessibility to RM strategies. Objectives: To assess the presence and biological potential of mesehchymal stem cells in three tissues (subchondral bone, synovial layer, periarticular adipose tissue) in late stages osteoarthritic patients. Material and Methods: Samples were collected from subjects undergoing total knee replacement (TKR). MSCs were isolated and cultured in complete αMEM with β FGF. Cell morphology and growth potential was assessed. Flow cytometry was used for detection of several relevant cell surface markers. Quantitative and qualitative assessment of differentiation potential towards three mesenchymal lineages (osteogenesis adipogenesis chondrogenesis) was performed. Time lapse life cell imaging of nondiferentiated cells over 24 hours period was used to determine cell kinetics. Results: Mesenchymal cells derived from all donors and tissue types showed morphology, growth and surface cell markers associated with stemness. All cell types underwent differentiation toward three mesenchymal lineages with significant differences between tissues of origin, not between donors. Cell kinetics, as derived from life imaging records, was variable with tissue of origin, significant higher for adipose derived MSCS. Conclusion: Human late stage OA mesenchymal tissues, contain progenitors with proliferative and differentiation potential of MSCs. These populations can be used for research and autologus regenerative therapies. Further comparative studies with age matched non OA samples has the potential of contributing to deepening knowledge about disease occurrence and progression.     

Subunit composition of the glycyl radical enzyme p-hydroxyphenylacetate decarboxylase. A small subunit, HpdC, is essential for catalytic activity.

p-Hydroxyphenylacetate decarboxylase from Clostridium difficile catalyses the decarboxylation of p-hydroxyphenylacetate to yield the cytotoxic compound p-cresol. The three genes encoding two subunits of the glycyl-radical enzyme and the activating enzyme have been cloned and expressed in Escherichia coli. The recombinant enzymes were used to reconstitute a catalytically functional system in vitro. In contrast with the decarboxylase purified from C. difficile, which was an almost inactive homo-dimeric protein (beta(2)), the recombinant enzyme was a hetero-octameric (beta(4)gamma(4)), catalytically competent complex, which was activated using endogenous activating enzyme from C. difficile or recombinant activating enzyme to a specific activity of 7 U.mg(-1). Preliminary results suggest that phosphorylation of the small subunit is responsible for the change of the oligomeric state. These data point to an essential function of the small subunit of the decarboxylase and may indicate unique regulatory properties of the system.