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J Clin Microbiol. 2009 Aug; 47(8): 2593–2595.
Published online 2009 Jul 1. doi:  10.1128/JCM.00367-09
PMCID: PMC2725657

Rapid and Simple Determination of Ciprofloxacin Resistance in Clinical Strains of Escherichia coli[down-pointing small open triangle]


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(6′)-Ib-cr (8-11).

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 diffusion-based 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 routinely grown in Luria-Bertani (LB) broth (1% Bacto tryptone, 0.5% yeast extract, 0.5% NaCl) or on LB agar at 37°C under aerobic conditions. The MICs of CIP were determined by automated microdilution (MicroScan Walkaway; Dade) and confirmed by Etest (AB Biodisk; Solna, Sweden) according to the manufacturer's instructions.


For molecular typing of the clinical strains of E. coli, a reaction with the primer pair comprising REP1 (5′-IIIGCGCCGICATCAGGC-3′) and REP2 (5′-ACGTCTTATCAGGCCTAC-3′) was used to amplify putative repetitive extragenic palindrome (REP)-like elements in the genomic bacterial DNA, as previously reported (1). Strains belonging to the same DNA group showed identical profiles or highly similar profiles (with up to two bands different).

Determination of DNA fragmentation.

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. Five milliliters of culture was then incubated with or without two different concentrations (0.1 and 1 μg/ml) of CIP (Sigma Aldrich, Madrid, Spain) for 40 min at 37°C. This was carried out as a blind procedure, without prior knowledge of the MICs. The two different concentrations of CIP were used for the following reasons. At 0.1 μg/ml, those bacterial isolates harboring some quinolone-resistant mechanism (which may act as a first step to yielding high CIP resistance through a second mechanism) were detectable at this low concentration. Although these isolates remain susceptible to CIP according to CLSI criteria (3), the clinical consequences of this altered CIP susceptibility remain unknown. On the other hand, a bacterial isolate with a CIP MIC higher than 1 μg/ml is considered intermediate (nonsusceptible) according to CLSI criteria (the CIP breakpoints were ≤1, 2, and ≥4 [susceptible, intermediate, and resistant, respectively]) and fully resistant (the CIP breakpoints were ≤0.5 and >1 [susceptible and resistant, respectively]) according to EUCAST criteria (http://www.srga.org/eucastwt/MICTAB/MICquinolones.htm).

Briefly, after CIP incubation, the cells were immersed in an agarose microgel on a slide, lysed, and stained with a fluorochrome in accordance with the manufacturer's instructions. CIP-induced DNA fragmentation is visualized as DNA spots that diffuse peripherally from the nucleoid. The greater the DNA fragmentation, the greater the number of DNA spots and the width of the diffusion zone around the residual nucleoid. The response to CIP-induced damage is quantified simply by measuring the widths of the diffusion zones of the DNA fragments emerging from the border of the nucleoid. Five different categories of DNA fragmentation were determined, according to the width of the diffusion zone: grade 0, no damage; grade I, low level of damage; grade II, intermediate damage; grade III, high level of damage; and grade IV, massive fragmentation (12).

Control strains.

The above-mentioned conditions were tested with three control strains of E. coli harboring specific mechanisms involved in reduced susceptibility to CIP (Fig. (Fig.1).1). E. coli strain J53 (CIP MIC of 0.012 μg/ml) showed massive fragmentation at 0.1 and 1 μg/ml of CIP (grade IV at each concentration); the same E. coli J53 strain harboring the previously reported plasmid-mediated quinolone-resistant determinant (9) (qnrA1) (CIP MIC of 0.19 μg/ml) showed a high level of fragmentation at 1 μg/ml (grade III) but no effect at 0.1 μg/ml of CIP. A very similar result was obtained with strain C-15 (CIP MIC of 0.25 μg/ml), which displayed an S83L replacement at GyrA (13). Nevertheless, the effect of 1 μg/ml CIP was much lower than in strain J53qnrA1, and only grade II fragmentation was achieved. Otherwise, E. coli clinical isolate C1341 (4) harboring replacements at GyrA (S83L and D87Y) and ParC (S80I) (CIP MIC of 4 μg/ml) showed no DNA fragmentation at either concentration of CIP, thus revealing the ability of the method to detect resistance to CIP and also subtle differences in degree of susceptibility to CIP.

FIG. 1.
Representative images of four strains of E. coli incubated with CIP for 40 min at doses of 0 μg/ml (a to d), 0.1 μg/ml (e to h), and 1 μg/ml (i to l); processed with the kit to determine chromosomal DNA fragmentation; and then ...

Results and discussion.

A total of 110 clinical strains of E. coli were initially selected. However, 15 out of 110 isolates were discounted due to a possible genetic relationship, as revealed by REP-PCR. Therefore, 95 isolates were subjected to further study of DNA fragmentation.

According to the MICs obtained by microdilution and CLSI criteria, 74 isolates were categorized as resistant to CIP (77.9%) and 21 were categorized as susceptible to CIP (22.1%). The DNA fragmentation assay correctly identified all 74 CIP-resistant strains. As regards the susceptible strains, all bacterial isolates were correctly assigned as susceptible to CIP (100% sensitivity and specificity). Moreover, in two cases, the DNA fragmentation assay was able to detect bacterial strains with a CIP MIC of 1 μg/ml and grade II to III DNA damage at 1 μg/ml but no damage (grade 0) at the lower concentration of CIP. Although these bacterial isolates are considered fully susceptible to CIP by CLSI criteria, they appeared to harbor some mechanism that operates at a low level, which may lead, at a later step, to a second mutation/mechanism and thus to high CIP resistance. The clinical impact of these strains with low levels of resistance to quinolones (or CIP) remains unknown. However, microbiology laboratories should report and alert clinicians to the presence of these isolates with reduced susceptibility to CIP.

The present findings are important in the field of antimicrobial resistance, as results can be obtained in 80 min (including the 40 min of CIP incubation) as regards the susceptibility or resistance to CIP of an E. coli clinical strain, compared with the 16 to 20 h needed to read a MIC to CIP by either microdilution or Etest. Preliminary results with five E. coli strains fully susceptible to CIP showed that when they were growing exponentially in LB broth culture rather than on LB agar, CIP sensitivity was immediately detected, without the need for prior incubation for 40 min. The presence of CIP during the 8 min required to prepare the microgel by mixing the aliquot of the culture with CIP and then with the liquid agarose, followed by cooling at 4°C in the freezer, was sufficient for visualization of CIP-induced DNA fragmentation.

In summary, the procedure (as a kit) reported here is a simple, fast, reproducible, and accurate method that enables routine determination of the susceptibility or resistance of clinical isolates of E. coli to CIP. Such rapid assessment may be of great interest for avoiding treatment failure in urinary tract infections and particularly in life-threatening infections, such as bacteremia, frequently caused by E. coli isolates. Further experiments are in progress to implement the technology in other combinations of antimicrobial agents and microorganisms of high clinical impact in order to detect multiresistant microorganisms in less than 2 hours.


This study was supported by the Spanish Network for Research on Infectious Diseases (REIPI), Instituto de Salud Carlos III (RD06/0008/0025); Fondo de Investigaciones Sanitarias (PI061368 and PI081613); and SERGAS (PS07/90) and grants from Xunta de Galicia (07CSA050916PR and INCITE07PXI916201ES).

We also thank L. Martínez, J. Vila, and C. Torres for the kind gift of control strains of E. coli.


[down-pointing small open triangle]Published ahead of print on 1 July 2009.


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