Fast Assessment of Resistance to Carbapenems and Ciprofloxacin of Clinical Strains of Acinetobacter baumannii

Bou, Germán; Otero, Fátima; Santiso, Rebeca; Tamayo, María; Fernández, María del Carmen; Tomás, María; Gosálvez, Jaime; Fernández, José Luis Fernández

doi:10.1128/jcm.01675-12

Cited by 21 publications

(16 citation statements)

References 23 publications

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“…Two variants of the Micromax kit for fluorescence microscopy (research use only) (Halotech DNA SL, Madrid, Spain), Micromax Q and Micromax WG, have been employed to evaluate the integrity of the nucleoid and of the cell wall, respectively. Their only difference lies in the composition of the lysing solution (27).…”

Section: Methodsmentioning

confidence: 99%

“…The slides were visualized under fluorescence microscopy and 200 to 500 bacteria were scored per experimental point. The scoring criterion of DNA fragmentation is the presence of diffused DNA fragments from the nucleoid (27,30). Otherwise, cell wall damage is evidenced when the nucleoid is released (27,28).…”

Section: Methodsmentioning

confidence: 99%

“…The scoring criterion of DNA fragmentation is the presence of diffused DNA fragments from the nucleoid (27,30). Otherwise, cell wall damage is evidenced when the nucleoid is released (27,28). The time required for the assay included 1 h 30 min of incubation of the bacteria in Mueller-Hinton broth, 1 h of incubation with colistin, 40 min of technical processing (10 min of microgel enclosing, 5 min of lysis, 15 min of washing and dehydrating, and 10 min of drying and staining), and 5 to 15 min of scoring under the microscope, for a total of 3 h 15 min to 3 h 30 min.…”

Section: Methodsmentioning

confidence: 99%

“…In a previously reported study, a simple procedure for the rapid assessment of resistance to carbapenems and ciprofloxacin in A. baumannii using Micromax technology was validated (27). In the case of carbapenems, cells immersed in a microgel on a slide were incubated with a lysing solution that removes the cell wall only in those bacteria in which peptidoglycan synthesis has been affected by the beta-lactam.…”

mentioning

confidence: 99%

“…Thus, only susceptible bacteria release the nucleoid, which is stained and visualized under fluorescence microscopy. Unaffected bacteria retain their standard shape (27,28). For ciprofloxacin assessment, the lysis procedure removes the cell wall in all bacteria.…”

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confidence: 99%

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Rapid Determination of Colistin Resistance in Clinical Strains of Acinetobacter baumannii by Use of the Micromax Assay

et al. 2013

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Colistin is an old antibiotic which has been used as a therapeutic option for carbapenem-and multidrug-resistant Gram-negative bacteria, like Acinetobacter baumannii. This pathogen produces life-threatening infections, mainly in patients admitted to intensive care units. Rapid detection of resistance to colistin may improve patient outcomes and prevent the spread of resistance. For this purpose, Micromax technology was evaluated in four isogenic A. baumannii strains with known mechanisms of resistance to colistin and in 66 isolates (50 susceptible and 16 resistant). Two parameters were determined, DNA fragmentation and cell wall damage. To assess DNA fragmentation, cells trapped in a microgel were incubated with a lysing solution to remove the cell wall, and the released nucleoids were visualized under fluorescence microscopy. Fragmented DNA was observed as spots that diffuse from the nucleoid. To assess cell wall integrity, cells were incubated with a lysis solution which removes only weakened cell walls, resulting in nucleoid release exclusively in affected cells. A dose-response relationship was demonstrated between colistin concentrations and the percentages of bacteria with DNA fragmentation and cell wall damage, antibiotic effects that were delayed and less frequent in resistant strains. Receiver operating characteristic (ROC) curves demonstrated that both DNA fragmentation and cell wall damage were excellent parameters for identifying resistant strains. Obtaining <11% of bacteria with cell wall damage after incubation with 0.5 g/ml colistin identified resistant strains of A. baumannii with 100% sensitivity and 96% specificity. Results were obtained in 3 h 30 min. This is a simple, rapid, and accurate assay for detecting colistin resistance in A. baumannii, with strong potential value in critical clinical situations.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Rapid Determination of Colistin Resistance in Clinical Strains of Acinetobacter baumannii by Use of the Micromax Assay

et al. 2013

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Rapid Detection of Bacterial Susceptibility or Resistance to Quinolones

Otero

Santiso

Tamayo

et al. 2017

Fast Detection of DNA Damage

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The emergence of multidrug resistant microorganisms together with the decline in discovery and development of new antibiotics is of great concern in health-care policy. In this alarming context, an early and well-tailored antibiotic therapy is a relevant strategy not only to improve clinical outcome but also to avoid appearance and spreading of perilous resistant strains. One of the most common antibiotic classes is fluoroquinolones. They trap the DNA girase and/or topoisomerase IV on the DNA, resulting in DNA fragmentation. We have developed the Micromax assay to determine, in situ, the integrity of the chromosomal DNA-nucleoid from microorganisms. This was validated as a simple procedure for the rapid assessment of the susceptibility or resistance to quinolones in gram-negative bacteria. After incubating with the quinolone, cells are trapped in an agarose microgel on a slide and incubated with a specific lysing solution to remove the cell wall and visualize the nucleoids under fluorescence microscopy. If the strain is susceptible to the quinolone, the bacterial nucleoids show a halo of diffusing DNA spots of fragmented DNA, whereas they appear intact in the resistant strain. The technical processing is performed in 40 min with practically total sensitivity and specificity.

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Phenotypic antibiotic susceptibility testing of pathogenic bacteria using photonic readout methods: recent achievements and impact

Tannert

Grohs

Popp

et al. 2018

Appl Microbiol Biotechnol

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Fast Assessment of Resistance to Carbapenems and Ciprofloxacin of Clinical Strains of Acinetobacter baumannii

Cited by 21 publications

References 23 publications

Rapid Determination of Colistin Resistance in Clinical Strains of Acinetobacter baumannii by Use of the Micromax Assay

Rapid Determination of Colistin Resistance in Clinical Strains of Acinetobacter baumannii by Use of the Micromax Assay

Rapid Detection of Bacterial Susceptibility or Resistance to Quinolones

Phenotypic antibiotic susceptibility testing of pathogenic bacteria using photonic readout methods: recent achievements and impact

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