DNA Fragmentation in Microorganisms Assessed In Situ

Fernández, José Luís; Cartelle, Mónica; Muriel, Lourdes; Santiso, Rebeca; Tamayo, María; Goyanes, Vicente; Gosálvez, Jaime; Bou, Germán

doi:10.1128/aem.00318-08

Cited by 37 publications

(38 citation statements)

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“…Because of the high ciprofloxacin concentrations used, cellular repair mechanisms were not able to increase DNA synthesis 26 and, therefore, a complete inhibition of DNA synthesis is observed. Furthermore, the lower fluorescence intensities obtained and higher fluorescence broadening without visible peaks could be related to DNA fragmentation, 27 because DNA fragments have a compromised ability to be stained with intercalating dyes, 24 such as DCR, resulting in lower relative DCR fluorescence. These results were further supported by microscopic images, which showed a low staining level and the inability of visualizing chromosomes.…”

Section: Discussionmentioning

confidence: 99%

Bacteriostatic versus bactericidal activity of ciprofloxacin in Escherichia coli assessed by flow cytometry using a novel far-red dye

et al. 2011

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As common microbiological methods for the assessment of bacteriostatic or bactericidal activities are very time-consuming, in this work we describe that the use of a novel far-red fluorescent stain, Vybrant DyeCycle Ruby (DCR) for the flow cytometric analysis of fluoroquinolone (ciprofloxacin) bacteriostatic and bactericidal activities in Escherichia coli proved to be specific for bacterial DNA and, after ciprofloxacin exposure, DNA distribution analysis was achieved using a 7.5 lM DCR concentration to stain 5Â10 5 ethanol-fixed bacterial cells. The analysis of the bacterial DNA histograms obtained from the ciprofloxacin concentrations tested, enabled the distinction between ciprofloxacin bacteriostatic and bactericidal activities.

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Section: Discussionmentioning

confidence: 99%

Bacteriostatic versus bactericidal activity of ciprofloxacin in Escherichia coli assessed by flow cytometry using a novel far-red dye

et al. 2011

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“…A Micro-Halomax kit for fluorescence microscopy (Halotech DNA SL, Madrid, Spain) was used to detect DNA fragmentation cell by cell, as previously reported (7). The clinical strains of E. coli were grown overnight on LB agar plates and then resuspended in LB broth at an optical density at 600 nm of 0.05.…”

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Rapid and Simple Determination of Ciprofloxacin Resistance in Clinical Strains of Escherichia coli

et al. 2009

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We recently reported a simple new in situ diffusion assay, developed as a kit, to visualize DNA fragmentation in single bacterial cells. Use of this assay in a collection of 95 genetically unrelated Escherichia coli clinical strains resulted in correct identification of all of the isolates as resistant or susceptible to ciprofloxacin, consistent with the MIC results. This relevant information is obtained in 80 min.Fluoroquinolones are among the most frequently prescribed antimicrobial agents worldwide, with ciprofloxacin (CIP) being one of the most widely used. The activity of these agents is mediated by the production of DNA fragmentation through trapping of DNA gyrase and/or topoisomerase IV on bacterial DNA (6). Fluoroquinolone MICs are mainly increased by mutations in the genes encoding the protein targets DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) but also by increasing the levels of specific efflux pumps, such as AcrAB-TolC, or by the presence of plasmid-mediated mechanisms, such as QnrA, QnrB, QnR, or the recently described aminoglycoside resistance gene aac(6Ј)- .Escherichia coli causes almost 50% of community-acquired urinary infections. Treatment with fluoroquinolones is recommended in such cases, especially in areas with predominant resistance to trimethoprim-sulfamethoxazole. However, over 22% of clinical isolates of E. coli may be resistant to CIP, and this tendency appears to be increasing (5,14). Resistance to CIP in clinical isolates of E. coli obtained from blood cultures has reached 19% in Spain. In a recent study, 43% of E. coli isolates from the normal intestinal microflora of humans were found to be resistant to quinolones (2).Given the alarming progressive increase in antibiotic resistance worldwide, it is of great importance to identify the resistant bacteria as soon as possible to avoid treatment failure and the spread of resistant microorganisms through misuse of antibiotics. Our research group recently developed a diffusionbased assay, developed as a kit, which enables straightforward and rapid (45 min) discrimination of bacteria with fragmented DNA. The kit can be used to evaluate DNA damage induced by antimicrobial agents. Since the activity of quinolones such as CIP depends on the production of fragmentation of bacterial DNA, we investigated the potential usefulness of the kit as a simple, fast procedure for detecting resistance to CIP in clinical situations by processing a collection of clinical strains of E. coli isolated from patients attending the Juan Canalejo Hospital.Bacterial strains, antibiotic susceptibility testing, and growth conditions. The procedure for determining chromosomal DNA fragmentation in situ was first assayed for control E. coli strains harboring known mechanisms of quinolone resistance as well as for 95 E. coli clinical strains isolated from outpatients (only 1 strain per patient was selected for further studies) attending the hospital in 2006 and 2007 and displaying different degrees of susceptibility to CIP. The E. coli isolates were r...

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“…Chromosomal DNA fragmentation is a hallmark of cell death both in eukaryotes during apoptosis and in prokaryotes in the presence of antibiotics (20,43). Another hallmark of PCD in bacteria is the production of ROS (20,36).…”

Section: Discussionmentioning

confidence: 99%

The Periodontal Pathogen Porphyromonas gingivalis Induces Expression of Transposases and Cell Death of Streptococcus mitis in a Biofilm Model

Duran-Pinedo¹,

Baker²,

Frias-Lopez

2014

Infect Immun

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Oral microbial communities are extremely complex biofilms with high numbers of bacterial species interacting with each other (and the host) to maintain homeostasis of the system. Disturbance in the oral microbiome homeostasis can lead to either caries or periodontitis, two of the most common human diseases. Periodontitis is a polymicrobial disease caused by the coordinated action of a complex microbial community, which results in inflammation of tissues that support the teeth. It is the most common cause of tooth loss among adults in the United States, and recent studies have suggested that it may increase the risk for systemic conditions such as cardiovascular diseases. In a recent series of papers, Hajishengallis and coworkers proposed the idea of the "keystone-pathogen" where low-abundance microbial pathogens (Porphyromonas gingivalis) can orchestrate inflammatory disease by turning a benign microbial community into a dysbiotic one. The exact mechanisms by which these pathogens reorganize the healthy oral microbiome are still unknown. In the present manuscript, we present results demonstrating that P. gingivalis induces S. mitis death and DNA fragmentation in an in vitro biofilm system. Moreover, we report here the induction of expression of multiple transposases in a Streptococcus mitis biofilm when the periodontopathogen P. gingivalis is present. Based on these results, we hypothesize that P. gingivalis induces S. mitis cell death by an unknown mechanism, shaping the oral microbiome to its advantage.T he human oral microbiome consists of over 600 individual taxa (1, 2). Its complexity and the fact that a large fraction of species is noncultivable have hampered our knowledge of the molecular and metabolic interactions that occur between the species of the bacterial biofilm and between bacteria and host.Periodontal disease is a polymicrobial inflammatory biofilm-mediated pathology that leads to a progressive loosening and eventual loss of teeth (3, 4), and it is responsible for half of all tooth loss in adults that occurs in moderate form in 39% of American adults and in severe form in 9% of adults. Polymicrobial diseases are increasingly being recognized as more frequent than previously thought (5). In these diseases there are complex interactions among the etiologic agents, which ultimately lead to disease. In the microbial etiology of periodontitis it is generally accepted that a consortium of bacteria, not a single microorganism, is involved in the disease. Nonetheless, it is well established that in destructive periodontitis, the "red complex" (Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) are key players in the disease process (6).A more holistic treatment of periodontitis or any other polymicrobial disease requires a better understanding of the ecology of the microbial community and the mechanisms that cause the shift from a mature stable biofilm to a dysbiotic community responsible for disease. Maintaining a healthy biofilm would be a less invasive strategy than removing an ...

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DNA Fragmentation in Microorganisms Assessed In Situ

Cited by 37 publications

References 37 publications

Bacteriostatic versus bactericidal activity of ciprofloxacin in Escherichia coli assessed by flow cytometry using a novel far-red dye

Bacteriostatic versus bactericidal activity of ciprofloxacin in Escherichia coli assessed by flow cytometry using a novel far-red dye

Rapid and Simple Determination of Ciprofloxacin Resistance in Clinical Strains of Escherichia coli

The Periodontal Pathogen Porphyromonas gingivalis Induces Expression of Transposases and Cell Death of Streptococcus mitis in a Biofilm Model

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