Real-time monitoring of Pseudomonas aeruginosa biofilm growth dynamics and persister cells' eradication.

Biofilm formation and the appearance of persister cells with low metabolic rates are key factors affecting conventional treatment failure and antibiotic resistance. Using impedance-based measurements, crystal violet staining and traditional culture we have studied the biofilm growth dynamics of 13 Pseudomonas aeruginosa strains under the effect of seven conventional antibiotics. Real-time growth quantifications revealed that the exposure of established P. aeruginosa biofilms to certain concentrations of ciprofloxacin, ceftazidime and tobramycin induced the emergence of persister cells, that showed different morphology and pigmentation, as well increased antibiotic resistance. Whole-genome sequencing of wildtype and persister cells identified several SNPs, a genomic inversion and a genomic duplication in one of the strains. However, these mutations were not uniquely associated with persisters, suggesting that the persistent phenotype may be related to metabolic and transcriptional changes. Given that mannitol has been proposed to activate bacterial metabolism, the synergistic combination of mannitol and ciprofloxacin was evaluated on clinical 48 h P. aeruginosa biofilms. When administered at doses ≥320 mg/L, mannitol was capable of preventing persister cell formation by efficiently activating dormant bacteria and making them susceptible to the antibiotic. These results were confirmed using viable colony counting. As the tested ciprofloxacin-mannitol combination appeared to fully eradicate mature biofilms, we conclude that impedance-based biofilm diagnostics, which permits antibiotic susceptibility testing and the identification of persister cells, is of great potential for the clinical practice and could aid in establishing treatment breakpoints for emerging biofilm-related infections.

Differential Effects of IGF-1R Small Molecule Tyrosine Kinase Inhibitors BMS-754807 and OSI-906 on Human Cancer Cell Lines.

We have determined the effects of the IGF-1R tyrosine kinase inhibitors BMS-754807 (BMS) and OSI-906 (OSI) on cell proliferation and cell-cycle phase distribution in human colon, pancreatic carcinoma, and glioblastoma cell lines and primary cultures. IGF-1R signaling was blocked by BMS and OSI at equivalent doses, although both inhibitors exhibited differential antiproliferative effects. In all pancreatic carcinoma cell lines tested, BMS exerted a strong antiproliferative effect, whereas OSI had a minimal effect. Similar results were obtained on glioblastoma primary cultures, where HGUE-GB-15, -16 and -17 displayed resistance to OSI effects, whereas they were inhibited in their proliferation by BMS. Differential effects of BMS and OSI were also observed in colon carcinoma cell lines. Both inhibitors also showed different effects on cell cycle phase distribution, BMS induced G2/M arrest followed by cell death, while OSI induced G1 arrest with no cell death. Both inhibitors also showed different effects on other protein kinases activities. Taken together, our results are indicative that BMS mainly acts through off-target effects exerted on other protein kinases. Given that BMS exhibits a potent antiproliferative effect, we believe that this compound could be useful for the treatment of different types of tumors independently of their IGF-1R activation status.

Cell Death Mechanisms Induced by CLytA-DAAO Chimeric Enzyme in Human Tumor Cell Lines.

The combination of the choline binding domain of the amidase N-acetylmuramoyl-L-alanine (CLytA)-D-amino acid oxidase (DAAO) (CLytA-DAAO) and D-Alanine induces cell death in several pancreatic and colorectal carcinoma and glioblastoma cell lines. In glioblastoma cell lines, CLytA-DAAO-induced cell death was inhibited by a pan-caspase inhibitor, suggesting a classical apoptotic cell death. Meanwhile, the cell death induced in pancreatic and colon carcinoma cell lines is some type of programmed necrosis. In this article, we studied the mechanisms that trigger CLytA-DAAO-induced cell death in pancreatic and colorectal carcinoma and glioblastoma cell lines and we acquire a further insight into the necrotic cell death induced in pancreatic and colorectal carcinoma cell lines. We have analyzed the intracellular calcium mobilization, mitochondrial membrane potential, PARP-1 participation and AIF translocation. Although the mitochondrial membrane depolarization plays a crucial role, our results suggest that CLytA-DAAO-induced cell death is context dependent. We have previously detected pancreatic and colorectal carcinoma cell lines (Hs766T and HT-29, respectively) that were resistant to CLytA-DAAO-induced cell death. In this study, we have examined the putative mechanism underlying the resistance in these cell lines, evaluating both detoxification mechanisms and the inflammatory and survival responses. Overall, our results provide a better understanding on the cell death mechanism induced by CLytA-DAAO, a promising therapy against cancer.

Effect of Dalbavancin on Staphylococcal Biofilms When Administered Alone or in Combination With Biofilm-Detaching Compounds.

Microorganisms grown in biofilms are more resistant to antimicrobial treatment and immune system attacks compared to their planktonic forms. In fact, infections caused by biofilm-forming Staphylococcus aureus and Staphylococcus epidermidis are a large threat for public health, including patients with medical devices. The aim of the current manuscript was to test the effect of dalbavancin, a recently developed lipoglycopeptide antibiotic, alone or in combination with compounds contributing to bacterial cell disaggregation, on staphylococcal biofilm formation and elimination. We used real-time impedance measurements in microtiter plates to study biofilm growth dynamics of S. aureus and S. epidermidis strains, in the absence or presence of dalbavancin, linezolid, vancomycin, cloxacillin, and rifampicin. Further experiments were undertaken to check whether biofilm-detaching compounds such as N-acetylcysteine (NAC) and ficin could enhance dalbavancin efficiency. Real-time dose-response experiments showed that dalbavancin is a highly effective antimicrobial, preventing staphylococcal biofilm formation at low concentrations. Minimum biofilm inhibitory concentrations were up to 22 higher compared to standard E-test values. Dalbavancin was the only antimicrobial that could halt new biofilm formation on established biofilms compared to the other four antibiotics. The addition of NAC decreased dalbavancin efficacy while the combination of dalbavancin with ficin was more efficient than antibiotic alone in preventing growth once the biofilm was established. Results were confirmed by classical biofilm quantification methods such as crystal violet (CV) staining and viable colony counting. Thus, our data support the use of dalbavancin as a promising antimicrobial to treat biofilm-related infections. Our data also highlight that synergistic and antagonistic effects between antibiotics and biofilm-detaching compounds should be carefully tested in order to achieve an efficient treatment that could prevent both biofilm formation and disruption.

CLytA-DAAO, Free and Immobilized in Magnetic Nanoparticles, induces Cell Death in Human Cancer Cells.

D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids generating hydrogen peroxide, a potential producer of reactive oxygen species. In this study, we used a CLytA-DAAO chimera, both free and bound to magnetic nanoparticles, against colon carcinoma, pancreatic adenocarcinoma, and glioblastoma cell lines. We found that the enzyme induces cell death in most of the cell lines tested and its efficiency increases significantly when it is immobilized in nanoparticles. We also tested this enzyme therapy in non-tumor cells, and we found that there is not cell death induction, or it is significantly lower than in tumor cells. The mechanism triggering cell death is apparently a classical apoptosis pathway in the glioblastoma cell lines, while in colon and pancreatic carcinoma cell lines, CLytA-DAAO-induced cell death is a necrosis. Our results constitute a proof of concept that an enzymatic therapy, based on magnetic nanoparticles-delivering CLytA-DAAO, could constitute a useful therapy against cancer and besides it could be used as an enhancer of other treatments such as epigenetic therapy, radiotherapy, and treatments based on DNA repair.