Cultures
Chromosomal DNA fragmentation in situ was assayed in the TG1 E. coli strain, which was grown routinely in Luria Bertani (LB) broth (1% Bacto-tryptone, 0.5% yeast extract, 0.5% NaCl) or on LB agar at 37°C in aerobic conditions. E. coli TG1 [genotype: F traD36 LacI q (lacZ)M15] proAB/supE (hsdMmcrB)5(r km k McrB) thi (lac-proAB). Cell growth in liquid cultures was evaluated by monitoring turbidity at OD600 using a spectrophotometer (Unicam 8625, Cambridge, UK). The minimum inhibitory concentration (MIC) was determined using the E-test (AB Biodisk, Solna Sweden) according to manufacturer's instructions. Viability was determined by colony counting after sequential dilutions and plating. To determine the percentage of viable cells, the number of cells seeded on the plate was counted using a cytometric camera.
Experiments
Three different experiments were performed with TG1 E. coli, all in triplicate. Typical experiments are presented. In the first, several colonies of TG1 E. coli were grown overnight on LB agar plates and then resuspended in LB broth at an OD600 of 0.05 and grown to an OD600 of 0.8. The colonies were then incubated with 0, 0.003, 0.006, 0.008, 0.012, 0.02, 0.04, 0.08, 0.1, 0.5, or 1 μg/ml CIP (Sigma) in 15 ml Falcon tubes containing 4 ml of LB broth for 40 min at 37°C with aeration and shaking, and then processed to measure the chromosomal DNA fragmentation.
In the second experiment, TG1 E. coli was removed from culture in LB agar, resuspended in LB broth at an OD600 of 0.5, and treated with 1 μg/ml CIP in LB broth at 37°C with aeration and shaking. Aliquots were removed after 0, 5, 10, 15, 20, 30, and 40 min of incubation, and processed to measure DNA fragmentation. The time needed to prepare the microgel with the cells enclosed, before the slide was immersed in the lysing solution, was 8 min (see next section). In the results, this time must be added to each incubation period. To complete this experiment, TG1 E. coli were cultured in liquid LB broth at 37°C for 23 h with aeration and shaking, and the growth was monitored by measuring the turbidity (OD600). The liquid cultures started at an OD600 of 0.05. Aliquots were removed during the exponentially growing phase at 3 h (i.e., at an OD600 of 0.52) and during the stationary phase at 7 h (OD600: 1.20), 9 h (OD600: 1.52) and 23 h (OD600: 1.84). At the end of each designated time, 1 μg/ml of CIP was added directly to the aliquot, and the aliquot was incubated at 37°C for 0 and 5 min, and then processed to measure the DNA fragmentation.
In the third experiment, the micro-organisms were grown overnight on LB agar plates, resuspended in LB broth at an OD600 of 0.05, grown to an OD600 of 0.8, and then incubated with 10, 1, or 0.1 μg/ml CIP in LB broth for 40 min at 37°C. After the incubation, the CIP was removed from the medium by centrifuging the bacteria and washing in plain LB broth. The bacteria were incubated at 37°C in LB broth with aeration and shaking, and aliquots were removed at 0, 1.5, 3, 4, 5, and 24 h. For the 0.1 μg/ml dose of CIP, the bacteria were also incubated for 6 h. One aliquot was used to measure the DNA fragmentation, and another was plated on LB agar at 37°C to measure the viability after 24 h of culture. Cultures without CIP and with CIP incorporated in the new LB medium added after washing after the initial CIP treatment were included and processed along with each dose and for the various incubation times.
Bacterial strains with low CIP sensitivity
Besides the experiments with TG1, DNA fragmentation was measured in four E. coli strains whose low sensitivity to CIP and underlying mechanisms are known. These included strains with mutations in the QRDR region from GyrA and ParC [16]. The isolates were C-15 (Ser83Leu from GyrA; CIP MIC = 0.25 μg/ml); 1273 (Ser83Leu and Asp87Tyr from GyrA; CIP MIC: 8.0 μg/ml), and 1383 (Ser83Leu and Asp87Tyr from GyrA together with Ser80Ile and Glu84Lys from ParC; CIP MIC: 128 μg/ml), and the control strain C-20 with no mutation in the QRDR region (CIP MIC: 0.007 μg/ml). The strain J53 with the plasmid-mediated quinolone-resistance gene qnrA1 (CIP MIC: 0.25 μg/ml) and its control strain J53 without the plasmid were also examined [17]. These strains were exposed to CIP at the MIC dose, at 10× and 100× the MIC dose, and at 0.5× and 0.25× the MIC dose for 40 min at 37°C in the exponentially growing phase, and DNA fragmentation was determined.
Determination of DNA fragmentation
The Micro-Halomax® kit for fluorescence microscopy (Halotech DNA SL, Madrid, Spain) was used. A thorough description has been published previously [15]. Essentially, an aliquot of each sample was diluted to a concentration of 5–10 million micro-organisms/ml in LB medium. The kit includes 0.5 ml snap cap microfuge tubes containing gelled aliquots of low-melting point agarose. The tube was placed in a water bath at 90–100°C for about 5 min to melt the agarose completely and then placed in a water bath at 37°C. Twenty-five microlitres of the diluted sample was added to the tube and mixed with the melted agarose. A 20 μl aliquot of the sample-agarose mixture was pipetted onto a precoated slide, and the sample was covered with a 22 mm × 22 mm coverslip. The slide was placed on a cold plate in the refrigerator (4°C) for 5 min to allow the agarose to produce a microgel with the trapped intact cells inside. The coverslip was removed gently, and the slide was immediately immersed horizontally in 10 ml of the lysing solution for 5 min at 37°C. The slide was washed horizontally in a tray with abundant distilled water for 3 min, dehydrated by incubating horizontally in cold (-20°C) ethanol of increasing concentration (70%, 90%, and 100%) for 3 min each, and air-dried in an oven.
The dried slide was incubated in a microwave oven at 750 W for 4 min, and the DNA was stained with 25 μl of the fluorochrome SYBR Gold (Molecular Probes, Eugene, OR, USA) diluted 1:100 in TBE buffer (0.09 M Tris-borate, 0.002M EDTA, pH 7.5) for 5 min in the dark.
Images were viewed under an epifluorescence microscope (Nikon E800), with a 100× objective and appropriate fluorescence filters, and the images were acquired using a high-sensitivity CCD camera (KX32ME, Apogee Instruments, Roseville, CA, USA). Groups of 16-bit digital images were obtained at each experimental time under similar conditions and stored as TIFF files. Image analysis was performed using a macro designed with Visilog 5.1 software (Noesis, Gif sur Yvette, France). This macro allows for thresholding and background subtraction, and delineates the circular area of diffusion of the DNA fragments from nucleoids. The width delimitated between the edge of the nucleoid and the circumference that limits the circular peripheral area of spreading of DNA fragments is the simplest parameter to estimate DNA fragmentation level after CIP treatment and was measured in μm. At each experimental time, 50–125 nucleoids were evaluated.
Statistical analysis
Because the data did not follow a normal distribution as ascertained by the Kolmogorov-Smirnov test, the non-parametric Mann-Whitney U test was performed to compare the groups. Significance was defined as P < 0.05.