Transfer of plasmids harbouring blaOXA-48-like carbapenemase genes in biofilm-growing Klebsiella pneumoniae: Effect of biocide exposure.

The spread of OXA-48-encoding plasmids from Klebsiella pneumoniae (OXA-48-Kpn), especially successful high-risk (HR) clones, is a growing concern. Biofilm formation can contribute to the dissemination of OXA-48-Kpn. It is not known whether biocides can affect the transfer of OXA-48-Kpn in biofilm. The aim of this study was to evaluate the effect of biocides on the conjugation frequency (CF) of OXA-48-Kpn in both biofilm and planktonic cultures. For that, seven OXA-48-Kpn isolates (4 belonging to HR clones and 3 to non-HR clones) were selected as donors. Each isolate was mixed (1:1) with Escherichia coli J53 (recipient) and grown on polystyrene microplates without biocides (control) and with 0.25x MIC of triclosan (TRI), chlorhexidine digluconate (CHX), povidone-iodine (POV), sodium hypochlorite (SOD) or ethanol (ETH). The CF was calculated as the number of transconjugants/number of E. coli J53. The results showed that for isolates growing in the absence of biocide, the mean fold change in the CF in biofilm with respect to that determined in planktonic cells (CF-BF/CF-PK) was 0.2 in non-HR isolates and ranged from 2.0 to 14.7 in HR isolates. In HR isolates grown in the presence of biocide, especially CHX, TRI, and ETH, the fold changes in CF-BF/CF-PK decreased, whereas in non-HR isolates the fold changes were similar or increased slightly with CHX, ETH, SOD and POV. In conclusion, the fold changes in the CF-BF/CF-PK are higher in HR isolates comparing to non-HR isolates in abscence of biocides. The fold changes in CF-BF/CF-PK of the HR isolates in the presence of biocides varied with the type of biocides, whereas in non-HR isolates, biocides have no significant effect, or produce only a slight increase in the fold change of CF-BF/CF-PK.

Role of Ca2+ in the secretory and biosynthetic response of porcine gonadotropes to substance P and gonadotropin-releasing hormone.

Substance P has been previously shown to stimulate luteinizing hormone (LH) secretion and synergistically enhance gonadotropin-releasing hormone (GnRH)-evoked LH release from cultured pig pituitary cells. To investigate the mechanisms involved in these responses, the effects of substance P (100 nM; 4 h) and/or GnRH (10 nM, 4 h) on LH release, LH intracellular content, and betaLH mRNA accumulation were evaluated in the absence or presence of extracellular Ca(2+). Likewise, the effects of substance P on the dynamics of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) were examined in single cells. Extracellular Ca(2+) deprivation abolished both substance P- and GnRH-stimulated LH release, as well as their synergistic interaction. The substance P antagonist D-Arg1,D-Phe5,-Trp7,9,Leu11-substance P (100 nM) blocked the stimulatory effect of substance P on LH release and its interaction with GnRH without affecting GnRH-induced LH secretion. Whereas substance P did not modify betaLH transcript levels, GnRH stimulated betaLH mRNA accumulation through a mechanism dependent upon extracellular Ca(2+). Substance P directly increased [Ca(2+)](i) in a 30% of gonadotropes by causing two distinct types of response kinetics with single-peak (predominant, 83.3%) or sustained-plateau profiles. Reduction of external [Ca(2+)] decreased by half the percent of responsive cells, which only showed single-peak profiles. Taken together, our results demonstrate that the ability of substance P to stimulate basal and GnRH-induced LH release is exerted directly upon gonadotropes, is extracellular Ca(2+)-dependent and does not seem to require net increases in betaLH mRNA levels. Moreover, [Ca(2+)](i) measurements revealed that although substance P action in pig gonadotropes is strongly dependent on extracellular Ca(2+) influx, it would also involve intracellular Ca(2+) mobilization. Finally, extracellular Ca(2+) also plays a requisite role to sustain GnRH-stimulated increases in both betaLH mRNA and LH release.