Sequential Hypertonic-Hypotonic Treatment Enhances Efficacy of Antibiotic against Acinetobacter baumannii Biofilm Communities

Infections with bacterial biofilm communities are highly tolerant of antibiotics. This protection is attributed, in part, to a hydrated extracellular polymeric substance (EPS) that surrounds the bacterial community and that limits antibiotic diffusion. In this study, we evaluated whether it is possible to dehydrate and then re-hydrate a biofilm as a means to increase antibiotic penetration and efficacy. Acinetobacter baumannii biofilms (24 h) were exposed to hypertonic concentrations of maltodextrin, sucrose or polyethylene glycol (PEG) as the dehydration step. These biofilms were then washed with deionized water containing 10 times the concentration of antibiotics needed to kill these bacteria in broth culture (50 µg/mL tobramycin, 300 µg/mL chloramphenicol, 20 µg/mL ciprofloxacin or 100 µg/mL erythromycin) as the rehydration step. Biofilms were then harvested, and the number of viable cells was determined. Sequential treatment with PEG and tobramycin reduced cell counts 4 to 7 log (p < 0.05) relative to combining PEG and tobramycin in a single treatment, and 3 to 7 log relative to tobramycin treatment alone (p < 0.05). Results were variable for other osmotic compounds and antibiotics depending on the concentrations used, likely related to mass and hydrophobicity. Our findings support future clinical evaluation of sequential regimens of hypertonic and hypotonic solutions to enhance antibiotic efficacy against chronic biofilm infections.

Optimization of the cytogenetic protocol for Pangasianodon hypophthalmus (Sauvage, 1878) and Clarias gariepinus (Burchell, 1822)

To obtain well spread chromosomes, the cytogenetic protocol for Pangasianodon hypophthalmus and Clarias gariepinus were optimized. This includes, the colchicine concentration (0.01%, 0.025%, 0.05%)/exposure duration (1, 3, and 5 h), hypotonic solution (distilled water or 0.075M KCl solution)/exposure duration (30 min, 1, and 2 h), the time of cell suspension preparation (at hypotonic treatment or before slide preparation) and chromosome aging period (0, 3, and 7 days in Carnoy’s fixative). In addition, the type (i.e., fin, gill or kidney) and the amount of tissue (10, 50, 100 or 150 mg) were also investigated. Regardless of the species, the result obtained showed that well-spread chromosomes could be obtained using the following optimized protocol: Juveniles are injected with 0.05% colchicine (at one ml kg−1) and allowed to swim for 3 h. Then, 50 mg of gill tissue is made into cell suspension in 0.075M KCl for 1 h. The cell suspension is treated in Carnoy’s fixative (changed three times at 20 min interval) and then aged for 3 days. Finally, chromosome slides are made and stained with 10% Giemsa for 1 h.

Efficacy of a Hypotonic Treatment for Peritoneal Dissemination from Gastric Cancer Cells: AnIn VivoEvaluation

The aim of the present study was to determine the efficacy of a hypotonic treatment for peritoneal dissemination from gastric cancer cells using anin vivomodel. We firstly evaluated the toxicity of a peritoneal injection of distilled water (DW) (2 mL for 3 days) in mice. Macroscopic and microscopic examinations revealed that the peritoneal injection of DW did not severely damage the abdominal organs of these mice. MKN45 gastric cancer cells preincubated with NaCl buffer or DW for 20 minutesin vitrowere then intraperitoneally injected into nude mice, and the development of dissemination nodules was analyzed. The total number, weight, and volume of the dissemination nodules were significantly decreased by the DW preincubation. We then determined whether the peritoneal injection of DW inhibited the establishment of peritoneal dissemination. After a peritoneal injection of MKN45 cells into nude mice, NaCl buffer or DW was injected into the abdominal cavity for 3 days. The total volume of dissemination nodules was significantly lower in DW-injected mice than in NaCl-injected mice. In conclusion, we demonstrated the safeness of a peritoneal injection of DW. Furthermore, the development of dissemination nodules from gastric cancer cells was prevented by a preincubation with or peritoneal injection of DW.

On the role of ATP release, ectoATPase activity, and extracellular ADP in the regulatory volume decrease of Huh-7 human hepatoma cells

Hypotonicity triggered in human hepatoma cells (Huh-7) the release of ATP and cell swelling, followed by volume regulatory decrease (RVD). We analyzed how the interaction between those processes modulates cell volume. Cells exposed to hypotonic medium swelled 1.5 times their basal volume. Swelling was followed by 41% RVD40 (extent of RVD after 40 min of maximum), whereas the concentration of extracellular ATP (ATPe) increased 10 times to a maximum value at 15 min. Exogenous apyrase (which removes di- and trinucleotides) did not alter RVD, whereas exogenous Na+-K+-ATPase (which converts ATP to ADP in the extracellular medium) enhanced RVD40 by 2.6 times, suggesting that hypotonic treatment alone produced a basal RVD, whereas extracellular ADP activated RVD to achieve complete volume regulation (i.e., RVD40 ≈100%). Under hypotonicity, addition of 2-(methylthio)adenosine 5′-diphosphate (2MetSADP; ADP analog) increased RVD to the same extent as exposure to Na+-K+-ATPase and the same analog did not stimulate RVD when coincubated with MRS2211, a blocker of ADP receptor P2Y13. RT-PCR and Western blot analysis confirmed the presence of P2Y13. Cells exhibited significant ectoATPase activity, which according to RT-PCR analysis can be assigned to ENTPDase2. Both carbenoxolone, a blocker of conductive ATP release, and brefeldin A, an inhibitor of exocytosis, were able to partially decrease ATPe accumulation, pointing to the presence of at least two mechanisms for ATP release. Thus, in Huh-7 cells, hypotonic treatment triggered the release of ATP. Conversion of ATPe to ADPe by ENTPDase 2 activity facilitates the accumulated ADPe to activate P2Y13 receptors, which mediate complete RVD.

Natronorubrum sediminis sp. nov., an archaeon isolated from a saline lake

Two novel haloalkaliphilic archaea, strains CG-6T and CG-4, were isolated from sediment of the hypersaline Lake Chagannor in Inner Mongolia, China. Cells of the two strains were pleomorphic, non-motile and strictly aerobic. They required at least 2.5 M NaCl for growth, with optimum growth at 3.4 M NaCl. They grew at pH 8.0–11.0, with optimum growth at pH 9.0. Hypotonic treatment with less than 1.5 M NaCl caused cell lysis. The two strains had similar polar lipid compositions, possessing C20C20 and C20C25 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. No glycolipids were detected. Comparison of 16S rRNA gene sequences and morphological features placed them in the genus Natronorubrum. 16S rRNA gene sequence similarities to strains of recognized species of the genus Natronorubrum were 96.2–93.8 %. Detailed phenotypic characterization and DNA–DNA hybridization studies revealed that the two strains belong to a novel species in the genus Natronorubrum, for which the name Natronorubrum sediminis sp. nov. is proposed; the type strain is CG-6T (=CECT 7487T =CGMCC 1.8981T =JCM 15982T).