The effect of photodynamic therapy using Radachlorin on biofilm-forming multidrug-resistant bacteria.

OBJECTIVES: This study aimed to test the effect of photodynamic therapy (PDT) on the inhibition and removal of biofilms containing multidrug-resistant Acinetobacter baumannii. METHODS: Using multidrug-resistant A. baumannii strains, an antibiotic susceptibility test was performed using the Gram-negative identification card of the Vitek 2 system (bioMérieux Inc., France), as well as an analysis of resistance genes, the effects of treatment with a light-emitting diode (LED) array using Radachlorin (RADA-PHARMA Co., Ltd., Russia), and transmission and scanning electron microscopy to confirm the biofilm-inhibitory effect of PDT. RESULTS: The antibiotic susceptibility test revealed multiple resistance to the antibiotics imipenem and meropenem in the carbapenem class. A class-D-type ß-lactamase was found, and OXA-23 and OXA-51 were found in 100% of 15 A. baumannii strains. After PDT using Radachlorin, morphological observations revealed an abnormal structure due to the loss of the cell membrane and extensive morphological changes, including low intracellular visibility and small vacuoles attached to the cell membrane. CONCLUSION: PDT involving a combination of LED and Radachlorin significantly eliminated the biofilm of multidrug-resistant A. baumannii. Observations made using electron microscopy showed that PDT combining LED and Radachlorin was effective. Additional studies on the effective elimination of biofilms containing multidrug-resistant bacteria are necessary, and we hope that a treatment method superior to sterilization with antibiotics will be developed in the future.

Hepatocyte-targeting single galactose-appended naphthalimide: a tool for intracellular thiol imaging in vivo.

We present the design, synthesis, spectroscopic properties, and biological evaluation of a single galactose-appended naphthalimide (1). Probe 1 is a multifunctional molecule that incorporates a thiol-specific cleavable disulfide bond, a masked phthalamide fluorophore, and a single galactose moiety as a hepatocyte-targeting unit. It constitutes a new type of targetable ligand for hepatic thiol imaging in living cells and animals. Confocal microscopic imaging experiments reveal that 1, but not the galactose-free control system 2, is preferentially taken up by HepG2 cells through galactose-targeted, ASGP-R-mediated endocytosis. Probe 1 displays a fluorescence emission feature at 540 nm that is induced by exposure to free endogenous thiols, most notably GSH. The liver-specificity of 1 was confirmed in vivo via use of a rat model. The potential utility of this probe in indicating pathogenic states and as a possible screening tool for agents that can manipulate oxidative stress was demonstrated in experiments wherein palmitate was used to induce lipotoxicity in HepG2 cells.

Rationally designed fluorescence 'turn-on' sensor for Cu2+.

A rationally designed, coumarin-based fluorescent sensor imino-coumarin (IC) displays high selectivity for Cu(2+) over a variety of competing metal ions in aqueous solution with a significant fluorescence increase. DFT/TDDFT calculations support that the fluorescence 'turn-on' of IC originates from blocking the electron transfer of the nitrogen lone pair upon complexation with Cu(2+). IC was successfully applied to microscopic imaging for detection of Cu(2+) in LLC-MK2 cells (in vitro) and several living organs (in vivo).

In vitro and in vivo photodynamic therapy of otitis media in gerbils.

OBJECTIVES/HYPOTHESIS: The aim of this study was to evaluate the antibacterial effects of photodynamic therapy (PDT) on common bacteria causing otitis media with effusion (OME). METHODS: An in vitro study was carried out using a hematoporphyrin derivative sensitizer (Photogem; Lemonosov Institute of Fine Chemical, Moscow, Russia) and a 632-nm diode laser on Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae. The presence of colony-forming units of the bacteria was examined, the microscopic structures of the bacteria were examined by transmission electron microscopy (TEM), and flow cytometry of the bacteria was performed. An in vivo PDT study was performed using gerbils. S. pneumoniae or H. influenzae were injected into bullae. The Photogem was injected into the bullae 2 days later when OME developed, and transcanal irradiation with the 632-nm diode laser (90 J) was performed. Middle ear and bulla were washed with Dulbecco's phosphate buffered saline (DPBS) and the washed DPBS was cultured. The presence of bacterial colonies was examined. RESULTS: The PDT was effective in killing all three kinds of bacteria. TEM showed damaged bacterial cell membranes and cytoplasmic structures, and the flow cytometry showed a lower number of viable bacteria in the PDT group compared to the control group. PDT was effective in killing S. pneumoniae in 87% of the infected bullae with OME, whereas it was effective in eradicating H. influenzae in 50% of the infected bullae with OME. CONCLUSIONS: The results of these studies demonstrated that PDT may be effective to treat otitis media. PDT may have clinical implications in the treatment of otitis media that is resistant to antibiotic therapy.

Coumarin-derived Cu(2+)-selective fluorescence sensor: synthesis, mechanisms, and applications in living cells.

A novel coumarin-based fluorogenic probe bearing the 2-picolyl unit (1) was developed as a fluorescent chemosensor with high selectivity and suitable affinity in biological systems toward Cu(2+) over other cations tested. The fluorescence on-off mechanism was studied by femtosecond time-resolved fluorescence (TRF) upconversion technique and ab initio calculations. The receptor can be applied to the monitoring of Cu(2+) ion in aqueous solution with a pH span 4-10. To confirm the suitability of 1 for biological applications, we also employed it for the fluorescence detection of the changes of intracellular Cu(2+) in cultured cells. The results indicate that 1 should be useful for the fluorescence microscopic imaging and the study on the biological functions of Cu(2+).

Chromofluorescent indicator for intracellular Zn2+/Hg2+ dynamic exchange.

The fluorescence of NABQ increases remarkably in the presence of Zn(2+) and is quenched by Hg(2+). As shown by confocal imaging, NABQ-Zn(2+) can penetrate cells, where the bound Zn(2+) is exchanged for Hg(2+). This results in the concomitant export of Hg(2+) from the cells, showing that NABQ can act as a Zn(2+) carrier and as a Hg(2+) extracting agent in living cells.

[Detection of rifampin resistant mycobacterium tuberculosis complex using denaturing HPLC].

BACKGROUND: Tuberculosis (TB) remains an important cause of morbidity and mortality throughout the world. The surge of TB has been accompanied by an increase in multi-drug-resistant tuberculosis (MDR-TB). In this study, we developed a denaturing HPLC (DHPLC) method for detecting rpoB gene mutation as a rifampin resistance based on sequence. METHODS: In this study, we used 99 mycobacterial isolates grown in Ogawa media. At first, we used a PCR method that can amplify the 235 bp and 136 bp rpoB DNAs of Mycobacterium tuberculosis complex (MTB) and Non-tuberculous mycobacteria (NTM). And then, PCR-restriction fragment length polymorphism (RFLP) of rpoB DNA (342 bp), which comprises the Rif(T) region, was used for the differential identification of Mycobacteria. Finally, we detected these amplicons by DHPLC, compared to PCR-RFLP results, and performed sequencing. RESULTS: Among 99 mycobacterial isolates, 80 (81%) were MTB and 19 (19%) were NTM. NTM were identified to 7 different species by DHPLC and PCR-RFLP. rpoB mutation was detected in 9 (11%) of the MTB specimens. These results were confirmed by using sequencing. CONCLUSIONS: DHPLC provided a rapid, simple, and automatable performance for detection of rifampin resistant Mycobacterium tuberculosis complex and would be helpful as a supplemental method in high-throughput clinical laboratories.