1 Department of Bacteriology, Mycology and Parasitology, 2 National Center of Antimicrobials and Infection Control and 3 Department of Virology, Statens Serum Institut, Copenhagen, Denmark
Received 8 July 2004; returned 27 September 2004; revised 1 December 2004; accepted 6 December 2004
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Abstract |
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Methods: The bladders were studied by use of colony counts, in situ hybridization and electron microscopy.
Results: The bacterial counts in the bladder remained 1034 cfu/bladder even after mecillinam treatment had finished, and re-growth in the urine was observed. In the bladder epithelium from treated mice, bacteria cells were occasionally seen, presumably representing intracellularly located bacteria.
Conclusions: This is the first in vivo study indicating that during mecillinam treatment E. coli cells can penetrate the mouse bladder epithelium and persist.
Keywords: UTIs , mouse models , antibiotic treatments
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Introduction |
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Only a few experiments have been performed in vivo on the persistence of bacteria in the bladder after antibiotic treatment. The purpose of this study was to directly examine the location of E. coli bacteria in the mouse bladder after treatment with mecillinam. At the end of treatment, the mice were followed for 2 weeks to find out whether bacteria surviving in the bladder re-established infection.
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Materials and methods |
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A type 1 fimbriated E. coli (strain 21623884-114), isolated from a patient with UTI, susceptible to mecillinam (MIC 0.5 mg/L) was used. The inoculation into the mouse bladder was performed on day 0 leaving the bladder with 5 x 1075 x 108 cfu.5 The animal experiments were conducted under the auspices of the Animal Experiments Inspectorate, the Danish Ministry of Justice.
Antibiotic treatment and sampling
Mecillinam solution (Selexid; Leo, Copenhagen, Denmark) was injected subcutaneously, at a dose of 0.5 mg/mouse twice a day, 6 h apart, for 3 days. The first injection was administered the day after inoculation. Further details about treatment and sampling are as previously described.5
Study A. Three mice, from the treated group and the control group, were sacrificed on day 1 4 h after one dose, on day 2 after two doses, on day, 6 h apart, 3 after four doses and on day 4 after six doses, respectively. Bacterial count determination was performed on the urine and kidneys. The bladders were fixed and studied by in situ hybridization and electron microscopy. Bacterial count determination was conducted on an additional three mice from each group (on day 4, 12 mice from each group).
Study B. For follow-up, six mice per group were sacrificed on days 5, 8, 12 (not control) and 18, and bacterial count determination was performed.
Bacterial count determination
The urine and organs were processed immediately after sampling, as described previously.5
Fixation of bladders
The bladder (of a killed mouse) was injected with 0.2 mL of Karnovsky fixative (KF) and after 15 min the bladder was isolated and further fixed for 60 min. The bladder was bisected lengthwise and fixed (KF) overnight at 4°C. One half of the bladder was dehydrated and embedded in paraffin for in situ hybridization, whereas the other half was prepared for ultrastructural studies (electron microscopy).
In situ hybridization
Sections, 5 µm thick, were mounted on glass slides and treated with xylene three times for 10 min, followed by 10 min of dehydration in 96% ethanol. After drying at room temperature, sections were circumscribed with a hydrophobic pen (Dako S2002; Dako, Glostrup, Denmark). Probe EC1531 (5'-CACCGTAGTGCCTCGTCATCA) specific for E. coli 23S rRNA, labelled with CY3, was used. Hybridizations were performed as previously described.6
Electron microscopy
The specimens were post-fixed in 1% OsO4 (w/v) in 0.1 M cacodylate buffer, pH 7.2, containing 0.01 M CaCl2 for 90 min, followed by en bloc staining with 2% (w/v) uranyl acetate in barbiturate buffer, pH 7.3, for 60 min. After dehydration and embedding in Epon (glycide ether 100; Merck, Darmstadt, Germany), 1 µm sections were stained with Toluidine Blue and areas of interest selected. Thin sections were stained with uranyl acetate and lead citrate. Electron microscopy was carried out using a Philips 201C electron microscope at 60 kV.
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Results |
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The course of the UTI is shown in Figure 1. In urine of the control group, 1067 cfu/mL remained even after 18 days, whereas the treated mice had sterile urine on day 5 followed by re-growth on day 8 up to 6 x 104 cfu/mL. The bacteriuria thereafter declined and disappeared after 18 days. The bladders of the control group showed continuous infections, with a median of 105 cfu/bladder. In the treated mice, the cfu decreased from 105 cfu/bladder after one dose to 103 cfu/bladder on day 4 after the sixth dose. At the end of mecillinam treatment, the bacterial counts in the bladder remained with a median of 1034 cfu/bladder even on day 18. In the kidneys of the control group, the amount of bacteria was 104 cfu/kidney on day 3 and declined slowly to sterile on day 18. The treated mice had infected kidneys on day 1 but were sterile on day 3 and the rest of the period.
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Bacterial cells were observed occasionally inside the bladder epithelium, presumably representing intracellularly located bacteria.
Electron microscopy
The ultrastructure of the bladder epithelium of the untreated mice disclosed a surface layer of cuboidal cells (Figure 2a and b), and a small number of bacteria were found in the lumen of the bladder. The organisms were localized close to the surface of the epithelium, and higher magnification disclosed bacteria with a characteristic Gram-negative cell wall (Figure 2a) lying close to the microvilli of the epithelial cell. In the mecillinam-treated mice, the surface epithelium had well developed microvilli (Figure 2c), and no bacteria were found in the lumen of the bladder. However, (after one mecillinam dose) organisms were found in the cytoplasm of superficial epithelial cells (Figure 2c and d). These cells had a number of vacuoles, thin cisternae and a round nucleus. The internalized bacteria had a Gram-negative cell wall structure (Figure 2d) and were surrounded by a membrane. No degenerative signs were seen in the organisms.
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Discussion |
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Rivers & Steck8
followed mice with E. coli UTI over a 2 month period, after 3 days of oral treatment with the combination trimethoprim/sulfamethoxazole (1:5). On day 14, they could still detect culturable bacteria in the bladder and urine, but after the 2 month period they only detected bacteriuria.8
It may be argued that the lack of bacterial eradication in the bladder is because the antibiotic concentration is too low. The mouse urinary concentration of trimethoprim/sulfamethoxazole is not stated in these reports and can therefore not be evaluated. We have previously measured the urine concentration of mecillinam in humans, and thereafter in treated mice, and adjusted the dose so the t > MIC (0.5 mg/L) was 67 h after one dose of 0.5 mg/mouse.5,11
The phenomenon of persistent bacterial counts in the bladder even after antibiotic treatment has been shown in vivo in the experimental mouse model after use of a range of antibiotic classes. As mecillinam and other ß-lactam antibiotics are primarily extracellular antibiotics it would be interesting to perform in vivo studies using intracellular antibiotics such as fluoroquinolones, tetracycline, trimethoprim or chloramphenicol. Clinical trials evaluating different mecillinam dosing regimens have shown that bacteriological cure rates approach or exceed 90%. The problem with these studies is that the follow-up period is usually 1014 days after the end of treatment, and as we can see in the mouse model the urinary sample tested at this time point is most often culture-negative. It is not known from the clinical studies whether there were bacteria in the bladder tissue and if these would later establish another UTI. The experimental UTI model in mice or other rodents is perfect for this kind of study. Further studies in the area are needed.
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References |
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