Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jul 20;13(7):676.
doi: 10.3390/antibiotics13070676.

Rapid and Simple Morphological Assay for Determination of Susceptibility/Resistance to Combined Ciprofloxacin and Ampicillin, Independently, in Escherichia coli

Isidoro López  1   2 , Fátima Otero  1   2 , María Del Carmen Fernández  3   4 , Germán Bou  3   4 , Jaime Gosálvez  5 , José Luis Fernández  1   2   3
Affiliations

Rapid and Simple Morphological Assay for Determination of Susceptibility/Resistance to Combined Ciprofloxacin and Ampicillin, Independently, in Escherichia coli

Isidoro López et al. Antibiotics (Basel). .

Abstract

Current antibiograms cannot discern the particular effect of a specific antibiotic when the bacteria are incubated with a mixture of antibiotics. To prove that this task is achievable, Escherichia coli strains were treated with ciprofloxacin for 45 min, immobilized on a slide and stained with SYBR Gold. In susceptible strains, the nucleoid relative surface started to decrease near the MIC, being progressively condensed as the dose increased. The shrinkage level correlated with the DNA fragmentation degree. Ciprofloxacin-resistant bacilli showed no change. Additionally, E. coli strains were incubated with ampicillin for 45 min and processed similarly. The ampicillin-susceptible strain revealed intercellular DNA fragments that increased with dose, unlike the resistant strain. Co-incubation with both antibiotics revealed that ampicillin did not modify the nucleoid condensation effect of ciprofloxacin, whereas the quinolone partially decreased the background of DNA fragments induced by ampicillin. Sixty clinical isolates, with different combinations of susceptibility-resistance to each antibiotic, were co-incubated with the EUCAST breakpoints of susceptibility of ciprofloxacin and ampicillin. The morphological assay correctly categorized all the strains for each antibiotic in 60 min, demonstrating the feasible independent evaluation of a mixture of quinolone and beta-lactam. The rapid phenotypic assay may shorten the incubation times and necessary microbial mass currently required for evaluation.

Keywords: DNA fragments; antibiotic combination; bacterial nucleoid; beta-lactam; quinolone; rapid antibiogram.

PubMed Disclaimer

Conflict of interest statement

J.G. and J.L.F. are advisers of Halotech DNA SL. All other authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Representative images of Escherichia coli TG1 strain incubated with increasing concentrations of ciprofloxacin (MIC: 0.012 mg/L) for 45 min and stained with SYBR Gold. The nucleoids were progressively condensed at the middle of the bacteria as the concentration of ciprofloxacin increased. The median relative nucleoid surface (RNS) with respect to the total surface of the bacteria was 46.9% (42.03–50.33) for 0 mg/L (a), 41.2% (37.85–44.38) for 0.012 mg/L (b), 28.7% (26.23–30.95) for 0.04 mg/L (c) and 13.4% (11.95–14.85) for 0.5 mg/L (d).
Figure 2
Figure 2
Relative nucleoid surface (RNS) of E. coli TG1 strain incubated with increasing concentrations of ciprofloxacin (MIC: 0.012 mg/L) for 45 min. The data are presented as box and whisker plots. The horizontal line in the box indicates the median, the lower line of the box is the first quartile, the upper line of the box is the third quartile and the whiskers (the end of the vertical lines) are the maximum and minimum values. Abnormal values are presented as dots outside the box.
Figure 3
Figure 3
Relative nucleoid surface (RNS) of E. coli strains with MIC 0.007, 0.012, 0.25 and 8 mg/L, incubated with ciprofloxacin at concentrations that are multiples of MIC.
Figure 4
Figure 4
Images of intercellular DNA fragments after incubation for 45 min with increasing concentrations of ampicillin in a susceptible E. coli strain (MIC: 4 mg/L). The median DNA fragments per µm2 were 0.03 (0.007–0.056) for 0 mg/L (a), 0.57 (0.42–0.75) for 2 mg/L (b), 1.40 (0.89–1.59) for 4 mg/L (c) and 2.28 (2.06–2.54) for 16 mg/L (d).
Figure 5
Figure 5
Intercellular DNA fragments per µm2 after incubation for 45 min with increasing concentrations of ampicillin in one susceptible E. coli strain (MIC: 4 mg/L) and one resistant (MIC: 128 mg/L). The horizontal line in the box indicates the median, the lower line of the box is the first quartile, the upper line of the box is the third quartile and the whiskers (the end of the vertical lines) are the maximum and minimum values. Abnormal and extreme abnormal values are presented as dots and asterisk, respectively, outside the box.
Figure 6
Figure 6
Evaluation of the influence of ampicillin on the relative nucleoid surface (RNS) effect of ciprofloxacin. One E. coli strain susceptible to ciprofloxacin (MIC: 0.047 mg/L) and ampicillin (MIC: 4 mg/L) was incubated with fixed concentrations of ciprofloxacin (0, 0.06, 0.25 or 0.5 mg/L) together with increasing concentrations of ampicillin (0, 1, 2, 4 and 8 mg/L). Numerical values are presented in Supplementary Table S1.
Figure 7
Figure 7
Evaluation of the influence of ciprofloxacin on the intercellular DNA fragments per µm2 produced by ampicillin (AMP). The E. coli strain susceptible to ciprofloxacin (MIC: 0.047 mg/L) and ampicillin (MIC: 4 mg/L) was incubated with fixed concentrations of ampicillin (0, 2, 4 or 8 mg/L) together with increasing concentrations of ciprofloxacin (0, 0.06, 0.125, 0.25, 0.5 and 1 mg/L). Numerical values are presented in Supplementary Table S2.
Figure 8
Figure 8
Representative images of the technique for the rapid detection of susceptibility or not to a quinolone and a beta-lactam simultaneously. Four E. coli strains were incubated with the EUCAST breakpoint concentrations of susceptibility for ciprofloxacin (0.25 mg/L) and ampicillin (8 mg/L), simultaneously for 45 min. The fluorescence microscopy images were obtained after technical processing. (a) Strain susceptible to both antibiotics (MIC ciprofloxacin: 0.012 mg/L; MIC ampicillin: 2 mg/L). The visualization of an intense central spot corresponding to a condensed nucleoid inside the bacteria is the marker of susceptibility to ciprofloxacin. The presence of a background of DNA fragments is indicative of susceptibility to ampicillin. (b) Strain non-susceptible to ciprofloxacin and susceptible to ampicillin (MIC ciprofloxacin: 8 mg/L; MIC ampicillin: 4 mg/L). The fluorescence from the nucleoid is spread inside the bacteria, and no central spot is evident. This is the pattern observed in the control E. coli, without quinolone treatment, thus evidencing resistance to the ciprofloxacin dose. Otherwise, the presence of background DNA fragments is the signal of susceptibility to ampicillin. (c) Strain susceptible to ciprofloxacin and non-susceptible to ampicillin (MIC ciprofloxacin: 0.047 mg/L; MIC ampicillin: 12 mg/L). The fluorescence inside the bacteria is concentrated in a central spot, being indicative of affectation by the quinolone. There is no background of DNA fragments, so the strain is resistant to the ampicillin dose. (d) Strain non-susceptible to both antibiotics (MIC ciprofloxacin: 4 mg/L; MIC ampicillin: 12 mg/L). The fluorescence from the nucleoid is spread inside the bacteria, so the strain is resistant to the ciprofloxacin dose. No background of DNA fragments is observed, so the strain is also resistant to the ampicillin dose.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Peleg A.Y., Hooper D.C. Hospital-acquired infections due to gram-negative bacteria. N. Engl. J. Med. 2010;362:1804–1813. doi: 10.1056/NEJMra0904124. - DOI - PMC - PubMed
    1. Progress on antibiotic resistance. Nature. 2018;562:307. doi: 10.1038/d41586-018-07031-7. - DOI - PubMed
    1. Cassini A., Högberg L.D., Plachouras D., Quattrocchi A., Hoxha A., Simonsen G.S., Colomb-Cotinat M., Kretzschmar M.E., Devleesschauwer B., Cecchini M., et al. Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015: A population-level modelling analysis. Lancet Infect. Dis. 2019;19:56–66. doi: 10.1016/S1473-3099(18)30605-4. - DOI - PMC - PubMed
    1. Davey P., Marwick C.A., Scott C.L., Charani E., McNeil K., Brown E., Gould I.M., Ramsay C.R., Michie S. Interventions to improve antibiotic prescribing practices for hospital in patients. Cochrane Database Syst. Rev. 2017;2:CD003543. doi: 10.1002/14651858.CD003543.pub4. - DOI - PMC - PubMed
    1. Jorgensen J.H., Ferraro M.J. Antimicrobial susceptibility testing: A review of general principles and contemporary practices. Clin. Infect. Dis. 2009;49:1749–1755. doi: 10.1086/647952. - DOI - PubMed

LinkOut - more resources