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1.  Molecular basis of the non-beta-lactamase-mediated resistance to beta-lactam antibiotics in strains of Haemophilus influenzae isolated in Canada. 
A study recently conducted across Canada showed that 64 of 2,503 clinical isolates of Haemophilus influenzae were resistant to beta-lactams without production of a beta-lactamase (L. D. Tremblay, J. L'Ecuyer, P. Provencher, M. G. Bergeron, and Canadian Study Group, Can. Med. Assoc. J. 143:895-900, 1990). The beta-lactamase-negative strains formed three distinct groups, with ampicillin MICs of 0.5 to 1, 2 to 4, and greater than or equal to 8 micrograms/ml for groups I, II, and III, respectively. We have investigated the mechanisms of resistance for eight strains originating from different infections and geographic areas. These strains were representative of groups I to III. Five strains were nontypeable, two were type B, and one was non-B. Chromosomal DNA extracted from each strain was used to transform the laboratory strain Rd. Transformants were selected on beta-lactam-containing plates and showed the same level of resistance to ampicillin as the donor strains. Differences in outer membrane proteins, porins, and lipopolysaccharide profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) did not change with resistance. Functional analyses of purified porins in artificial lipid bilayer experiments did not explain resistance. Peptidoglycan synthesis was measured by incorporation of [14C]alanine into trichloroacetic acid-insoluble cell wall material in the presence of chloramphenicol. The growth rate and the rate of peptidoglycan synthesis observed for the transformants of the isogenic set did not correlate with resistance. Whole-cell labeling with 125I-penicillin revealed modifications in penicillin-binding proteins (PBPs) among the transformants. In particular, PBPs 3A and 3B (65 and 63 kDa, respectively) showed a decrease in affinity for beta-lactams in all transformants (groups I, II, and III) and correlated with an increased MIC except in the transformant of group III, which showed higher levels of resistance. Partial purification and proteolytic digestion of 125I-penicillin-labeled PBP 3B led to two types of CnBr peptide profiles on SDS-PAGE, the profiles of the transformed strains from groups I and II being different from those of the control group and group III. Finally, electron microscopy revealed a distinct cell filamentation for the group III transformants. These data clearly indicate that changes in PBPs are a common mechanism that results in a significant level of non-beta-lactamase-mediated beta-lactam resistance in H. influenzae despite serotype, origin of isolation, or geographic distribution.
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PMCID: PMC191612  PMID: 1510447
2.  Attenuation by daptomycin of gentamicin-induced experimental nephrotoxicity. 
Previously, daptomycin was shown to reduce tobramycin nephrotoxicity in vivo (D. Beauchamp, M. Pellerin, P. Gourde, M. Pettigrew, and M. G. Bergeron, Antimicrob. Agents Chemother. 34:139-147, 1990; C. A. Wood, H. C. Finkbeiner, S. J. Kohlhepp, P. W. Kohnen, and D. C. Gilbert, Antimicrob. Agents Chemother. 33:1280-1285, 1989). Female Sprague-Dawley rats were treated with saline (NaCl, 0.9%), daptomycin (10 mg/kg of body weight every 12 h, subcutaneously), gentamicin (30 mg/kg/12 h, intraperitoneally) or with a combination of daptomycin plus gentamicin over a 10-day period. Animals were killed 4, 10, and 20 days after the end of treatment. Four days after the end of drug administration, gentamicin and daptomycin levels in the renal cortices of animals treated with the combination of daptomycin and gentamicin were significantly higher than in those of rats given gentamicin or daptomycin alone (P < 0.01). Despite the higher cortical concentrations of gentamicin, rats given the combination of gentamicin and daptomycin had less reduction in renal cortex sphingomyelinase activity, less evidence of regeneration of cellular cortical cells ([3H]thymidine incorporation into cortex DNA), lower creatinine concentration in serum, and less histopathologic evidence of injury than rats given gentamicin alone. By immunogold technique, both daptomycin and gentamicin were localized to the lysosomes of proximal tubular cells, regardless of whether animals received the drugs alone or in combination. Interestingly, myeloid body formation occurred in both those animals given gentamicin alone and those given daptomycin plus gentamicin. No significant changes were observed for all groups between 10 and 20 days after the end of therapy, suggesting that the toxicity of gentamicin was not delayed by the concomitant injection of daptomycin. The results confirm that daptomycin can attenuate experimental gentamicin nephrotoxicity.
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PMCID: PMC188145  PMID: 8067733
3.  Daptomycin may attenuate experimental tobramycin nephrotoxicity by electrostatic complexation to tobramycin. 
The lipopeptidic antibiotic daptomycin is reported to reduce experimental tobramycin nephrotoxicity (D. Beauchamp, M. Pellerin, P. Gourde, M. Pettigrew and M. G. Bergeron, Antimicrob. Agents Chemother. 34:139-147, 1990; C. A. Wood, H. C. Finkbeiner, S. J. Kohlhepp, P. W. Kohnen, and D. C. Gilbert, Antimicrob. Agents Chemother. 33:1280-1285, 1989). In an attempt to explain these results, the in vivo and in vitro interactions between daptomycin and tobramycin were studied. Tobramycin alone and preincubated with negatively charged phospholipid bilayers (liposomes) was dialyzed against increasing concentrations of daptomycin in buffer at pH 5.4. A significant drop in the concentration of tobramycin was observed when daptomycin was added to the opposite half cells. Furthermore, daptomycin induced a concentration-dependent release of lipid-bound tobramycin. Gold labeling experiments showed that daptomycin could be incorporated into phospholipid layers. Female Sprague-Dawley rats were treated with daptomycin alone, with tobramycin alone, or with the combination over 2 to 10 days. Levels of daptomycin and tobramycin in serum were similar in all groups. The levels of tobramycin in the renal cortex increased significantly with time and, on day 10, reached values of 654 +/- 122 and 844 +/- 298 micrograms/g of tissue (mean +/- standard deviation; not significant) in animals treated with tobramycin and the combination of daptomycin-tobramycin, respectively. No significant difference was observed in the levels of tobramycin in the kidneys between animals treated with tobramycin or the daptomycin-tobramycin combination at any time. By contrast, daptomycin levels were significantly higher in the renal cortexes of animals treated with daptomycin-tobramycin in comparison with those in the renal cortexes of animals treated with daptomycin alone on days 6,8, and 10 (P < 0.01). For immunogold labeling studies, animals were killed 4 h after a single injection of daptomycin alone or daptomycin in combination with tobramycin. Daptomycin was found throughout the matrixes of the lysosomes of proximal tubular cells of animals treated with daptomycin alone. In animals treated with the combination of daptomycin and tobramycin, daptomycin was associated with intralysosomal myeloid bodies. Our results suggest that daptomycin might attenuate experimental aminoglycoside nephrotoxicity by interacting with the aminoglycoside, perhaps electrostatically, and thereby protecting intracellular targets of toxicity.
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PMCID: PMC284536  PMID: 8031040
4.  Subcellular distribution of daptomycin given alone or with tobramycin in renal proximal tubular cells. 
Previous studies in experimental animals showed that daptomycin, a lipopeptide antibiotic, protects against aminoglycoside nephrotoxicity (C. A. Wood, H. C. Finkbeiner, S. J. Kohlhepp, P. W. Kohnen, and D. N. Gilbert, Antimicrob. Agents Chemother. 33:1280-1285, 1989; D. Beauchamp, M. Pellerin, P. Gourde, M. Pettigrew, and M. G. Bergeron, Antimicrob. Agents Chemother. 34:139-147, 1990). In order to better understand the mechanism involved in this protective effect, the subcellular distribution of daptomycin was investigated in the proximal tubular cells of animals treated with daptomycin alone or in combination with tobramycin. A first group of female Sprague-Dawley rats received a single intravenous injection of daptomycin at a dose of 100 mg/kg of body weight and were killed at 10 min, 1 h, or 24 h after the injection. Other groups of rats were treated during 10 days with saline (NaCl, 0.9%), tobramycin at dosages of 20 mg/kg/12 h, daptomycin at dosages of 10 mg/kg/12 h, or the combination tobramycin-daptomycin at the same dosages. At the time of sacrifice, the renal cortex of the right kidney of each animal was dissected, and small blocks of tissue were fixed, dehydrated, and embedded in Araldite 502 epoxy resin. The subcellular distribution of daptomycin and tobramycin was determined on ultrathin sections by immunogold labeling. Ten minutes after the injection of daptomycin alone, gold particles were seen over the brush border membrane and on the membranes of the endocytic vacuoles of proximal tubular cells. One hour after the injection, a similar distribution was seen and numerous gold particles were found over the lysosomes of proximal tubular cells. The results suggest that daptomycin might protect against aminoglycoside nephrotoxicity by interfering with the interaction between the aminoglycoside and phospholipids inside the lysosomes of proximal tubular cells.
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PMCID: PMC284424  PMID: 8192441
5.  Toward Automatic Label-Free Whispering Gallery Modes Biodetection with a Quantum Dot-Coated Microsphere Population 
Nanoscale Research Letters  2010;5(3):524-532.
We explore a new calibration-free approach to biodetection based on whispering gallery modes (WGMs) without a reference measure and relative shifts. Thus, the requirement to keep track of the sensor position is removed, and a freely moving population of fluorophore-doped polystyrene microspheres can now fulfill this role of sensing resonator. Breaking free from fixed surface-based biosensing promotes adhesion between the microsphere sensors and the analytes since both can now be thoroughly mixed. The 70-nm-wide spectrum of green fluorescent microbeads allows us to monitor over 20 WGMs simultaneously without needing evanescent light coupling into the microspheres, hence enabling remote sensing. Since the exact radius of each microsphere is unknown a priori, it requires algorithmic analyses to obtain a reliable result for the refractive index of a solution. We first test our approach with different solutions of alcohol in water obtaining 3 × 10−4 precision on the refractive index at lower concentrations. Then, the solutions of bacterial spores in water yield clear evidence of biodetection in the statistical analysis of WGMs from 50 microspheres. To extend the fluorescence spectral range of our WGM sensors, we present preliminary results on coating microspheres with CdSe/ZnS quantum dots.
doi:10.1007/s11671-010-9541-1
PMCID: PMC2894210  PMID: 20672075
Whispering gallery modes; Biodetection; Optical resonances; Morphology-dependant resonances; Refractometers; Microspheres; Sensors
6.  vanD and vanG-Like Gene Clusters in a Ruminococcus Species Isolated from Human Bowel Flora▿  
Antimicrobial Agents and Chemotherapy  2007;51(11):4111-4117.
A vancomycin-resistant, anaerobic, gram-positive coccus containing the vanD and vanG-like genes (strain CCRI-16110) was isolated from a human fecal specimen during a hospital surveillance program to detect carriers of vancomycin-resistant enterococci. Comparison of the 16S rRNA gene sequence of strain CCRI-16110 with databases revealed a potentially novel Ruminococcus species that was most similar (<94% identity) to Clostridium and Ruminococcus species. Strain CCRI-16110 was highly resistant to vancomycin and teicoplanin (MICs of >256 μg/ml). The complete DNA sequence of the vanD cluster was most similar (98.2% identity) to that of Enterococcus faecium BM4339, containing the vanD1 allele. An intD gene with 99% identity with that of this E. faecium strain was found to be associated with the vanD gene cluster of this novel anaerobic bacterium. Strain CCRI-16110 also harbors genes encoding putative VanSG, VanG, and VanTG proteins displaying 56, 73.6, and 55% amino acid sequence identity, respectively, compared to the corresponding proteins encoded by the vanG1 and vanG2 operons of Enterococcus faecalis BM4518 and N03-0233. This study reports for the first time an anaerobic bacterium containing the vanD gene cluster. This strain also harbors a partial vanG-like gene cluster. The presence of vanD- and vanG-containing anaerobic bacteria in the human bowel flora suggests that these bacteria may serve as a reservoir for the vanD and vanG vancomycin resistance genes.
doi:10.1128/AAC.00584-07
PMCID: PMC2151448  PMID: 17724150
7.  High Prevalence of Glycopeptide Resistance Genes vanB, vanD, and vanG Not Associated with Enterococci in Human Fecal Flora 
Antimicrobial Agents and Chemotherapy  2005;49(11):4784-4786.
The presence of Enterococcus-associated vancomycin resistance genes vanA, vanB, vanD, vanE, and vanG in rectal swabs was investigated in two hospitals using PCR. All vanA genes detected were associated with vancomycin-resistant enterococci (VRE), whereas VRE-associated vanB genes were detected in only one hospital (4.7%). However, in both hospitals, high prevalences of vanB (6.2 and 2.3%), vanD (43.8 and 26.7%), and vanG (10.5 and 6.9%) genes not associated with enterococci were found.
doi:10.1128/AAC.49.11.4784-4786.2005
PMCID: PMC1280134  PMID: 16251331
8.  Tissue penetration of ciprofloxacin after single and multiple doses. 
The pharmacokinetics and the suction-induced blister fluid penetration of ciprofloxacin were compared after a single dose (500 mg) and after multiple dosing (500 mg q8h for 13 doses). Significantly higher peak levels of ciprofloxacin in serum were observed after multiple dosing (3.51 versus 2.26 micrograms/ml; P less than 0.01). Increased elimination half-life occurred after multiple dosing; this seems to be mostly related to decreased systemic clearance secondary to a diminished nonrenal clearance (240.0 versus 125.0 ml/min). Ciprofloxacin appeared rapidly in the blister fluid, and the percentage of penetration (AUC0-tBF/AUC0-t serum) yielded values of 88.8 versus 84.7% after single and multiple doses, respectively. Ciprofloxacin levels in serum and blister fluid at the end of the dosing interval (8 h) were superior or almost superior to MICs for sensitive organisms including Pseudomonas aeruginosa. Comparative studies of ciprofloxacin metabolite excretion after multiple doses in healthy subjects and in renal insufficiency patients are needed.
PMCID: PMC180421  PMID: 2940974
9.  New Real-Time PCR Assay for Rapid Detection of Methicillin- Resistant Staphylococcus aureus Directly from Specimens Containing a Mixture of Staphylococci 
Journal of Clinical Microbiology  2004;42(5):1875-1884.
Molecular methods for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) are generally based on the detection of an S. aureus-specific gene target and the mecA gene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) and S. aureus, either of which can carry mecA. In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosome mec (SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptible S. aureus (MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was ∼25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.
doi:10.1128/JCM.42.5.1875-1884.2004
PMCID: PMC404602  PMID: 15131143
10.  Differential distributions in tissues and efficacies of aztreonam and ceftazidime and in vivo bacterial morphological changes following treatment. 
The differential tissue distributions of aztreonam and ceftazidime within fibrin clots infected with Pseudomonas aeruginosa, Enterobacter cloacae, and Serratia marcescens, their efficacies, and the in vivo bacterial morphological changes induced by these drugs were evaluated. Rabbits were given intravenously a single dose of 100 mg of either agents/kg of body weight. In the cores of the clots, the peak levels of both drugs were much lower than those observed in the peripheries and in serum. Aztreonam's half-lives within the peripheries and in the cores of the fibrin clots were up to six times higher than observed in serum, while ceftazidime's half-lives in clots were twice that observed in serum. This resulted in a much greater penetration ratio for aztreonam than for ceftazidime. Both drugs controlled the growth of P. aeruginosa in vivo, but E. cloacae and S. marcescens responded better to ceftazidime. Morphological changes were more abundant in the peripheries than in the cores of the clots. In the control group, P. aeruginosa's morphology in the cores was different than that in the peripheries of the clots. Against P. aeruginosa, aztreonam did induce morphological changes in the cores while ceftazidime did not. Electron microscopic studies revealed that morphological changes associated with aztreonam seemed different than those of ceftazidime. Along with elongation of bacteria, more bow tie and herniated bacteria were observed with aztreonam. Though both agents selectively affect PBP 3, as manifested by elongated bacteria, they induce in the peripheries of the clots thickening, breaks, and detachment in bacterial cell walls, alterations which are generally associated with antibiotics affecting PBP 1a and 1b.
PMCID: PMC163720  PMID: 9021198
11.  Time-restricted feeding schedules modify temporal variation of gentamicin experimental nephrotoxicity. 
The effect of timing of gentamicin dosing relative to food access periods was evaluated in experimental animals. Female Sprague-Dawley rats were treated for 4 and 10 days with gentamicin (40 mg/kg of body weight/day) intraperitoneally at either 0700, 1300, 1900, or 0100 h according to three food presentation schedules: food was available from 0800 to 1600 h in the first group, from 1600 to 0000 h in the second group, and from 0000 to 0800 h in the last group. Animals were thus subjected to a restricted feeding period. Results indicate that time-restricted feeding schedules displace the peak and the trough of gentamicin-induced renal toxicity, as evaluated by changes in the inhibition of sphingomyelinase activity, cellular regeneration (incorporation of [3H]thymidine into DNA of renal cortex), and blood urea nitrogen and serum creatinine levels, as well as histopathological lesions observed after 10 days of treatment. In fact, the toxicity was minimal when gentamicin was injected during the feeding period, while the maximal toxicity was found when gentamicin was administered during the fasting period. It is concluded that the feeding period can modulate aminoglycoside nephrotoxicity. The time of dosing of gentamicin relative to the time of feeding seems to be a more important modulator of gentamicin nephrotoxicity than the light-dark cycle.
PMCID: PMC163942  PMID: 9210668
12.  Attenuation of gentamicin-induced nephrotoxicity in rats by fleroxacin. 
The effect of fleroxacin on gentamicin-induced nephrotoxicity was evaluated with female Sprague-Dawley rats. Animals were injected during 4 or 10 days with saline (NaCl; 0.9%), gentamicin alone at doses of 10 and 40 mg/kg of body weight/12 h (subcutaneously), fleroxacin alone at a dose of 25 mg/kg/12 h (intraperitoneally), or the combination gentamicin-fleroxacin in the same regimen. Gentamicin induced a dose- and time-dependent renal toxicity as evaluated by gentamicin cortical levels, sphingomyelinase activity in the renal cortex, histopathologic and morphometric analysis, blood urea nitrogen and serum creatinine levels, and cellular regeneration ([3H]thymidine incorporation into DNA of cortical cells). The extent of these changes was significantly reduced when gentamicin was given in combination with fleroxacin. Although the mechanisms by which fleroxacin reduces the nephrotoxic potential of gentamicin are unknown, we propose that the fleroxacin-gentamicin combination enhances exocytosis activity in proximal tubular cells, as suggested by the higher excretion of urinary enzymes and lower cortical levels of gentamicin observed in animals treated with the combination fleroxacin-gentamicin compared with those treated with gentamicin alone. The protective effect of fleroxacin on gentamicin nephrotoxicity should be investigated further.
PMCID: PMC163893  PMID: 9174177
13.  Association of nitric oxide production by kidney proximal tubular cells in response to lipopolysaccharide and cytokines with cellular damage. 
Recent findings suggest that nitric oxide (NO) is an important biologic mediator which exerts a wide variety of effects on numerous physiological and pathophysiological processes. L-Arginine is oxidized to L-citrulline with concomitant NO production; as a result, nitrate and nitrite accumulates. This study was conducted to determine the potential NO production by proximal tubular cells (PTC) in response to bacterial lipopolysac-charides (LPS) and cytokines and to evaluate the cytotoxic effect associated with NO release. After a 7-day stimulation with LPS (100 micrograms/ml), interleukin-1 beta (IL-1 beta) (10 ng/ml), and tumor necrosis factor alpha (TNF-alpha) (10 ng/ml), the nitrate and nitrite levels were determined by a spectrophotometric method based on the Griess reaction. Moreover, alpha-methylglucopyranoside phosphate and lactate dehydrogenase release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay served as indicators of sodium-dependent hexose transport integrity and cell death, respectively. IL-1 beta and TNF-alpha used alone or together or combined with LPS led to a significant generation of NO by PTC. Our results also demonstrate that NO induced by LPS and cytokines could inhibit sodium-dependent transport and could induce PTC damage.
PMCID: PMC163750  PMID: 9055992
14.  Quantitation of cytomegalovirus (CMV) DNA in leukocytes of human immunodeficiency virus-infected subjects with and without CMV disease by using PCR and the SHARP Signal Detection System. 
Journal of Clinical Microbiology  1997;35(2):525-526.
We report the development of a simple and rapid PCR assay for quantitation of the cytomegalovirus (CMV) DNA load in polymorphonuclear leukocytes. Using this system, a very good correlation was found between a high number of CMV copies in the blood and the presence of CMV disease in subjects with AIDS.
PMCID: PMC229619  PMID: 9003635
15.  Species-specific and ubiquitous DNA-based assays for rapid identification of Staphylococcus epidermidis. 
Journal of Clinical Microbiology  1996;34(12):2888-2893.
Staphylococcus epidermidis is an aerobic gram-positive coccus that is now recognized among the coagulase-negative staphylococci as an etiological agent with an important range of pathogenicity in humans. Several diagnostic kits based on biochemical or immunological reactions can efficiently identify Staphylococcus aureus. However, these tests are often unreliable for the identification of coagulase-negative staphylococcal species including S. epidermidis. Since DNA-based assays for the species-specific identification of S. epidermidis remain unavailable, we have developed such tests in order to improve the accuracy and the rapidity of tests for the diagnosis of S. epidermidis infections. On the basis of the results of hybridization assays with clones randomly selected from an S. epidermidis genomic library, we identified a chromosomal DNA fragment which is specific and 100% ubiquitous for the identification of S. epidermidis. This 705-bp fragment was sequenced and used to design PCR amplification primers. PCR assays with the selected primers were also highly specific and ubiquitous for the identification from bacterial cultures of clinical isolates of S. epidermidis from a variety of anatomic sites. While three strains of S. capitis were misidentified as S. epidermidis with the API Staph-Ident system and 2.5% of the S. epidermidis identifications were inconclusive with the MicroScan Autoscan-4 system, the PCR assay was highly specific and allowed for the correct identification of all 79 S. epidermidis strains tested. The PCR assays developed are simple and can be performed in about 1 h. The DNA-based tests provide novel diagnostic tools for improving the diagnosis of S. epidermidis infections.
PMCID: PMC229428  PMID: 8940417
16.  Effects of fasting on temporal variations in nephrotoxicity of gentamicin in rats. 
Evidence for temporal variations in the nephrotoxicity of low doses of aminoglycosides were recently shown by using specific and sensitive parameters of renal toxicity. The aim of the present study was to evaluate the effect of a short period of fasting on the temporal variations in the renal toxicity of gentamicin. Twenty-eight normally fed (i.e., food and water were available ad libitum throughout the experiment) female Sprague-Dawley rats (weight, 175 to 220 g) and 28 fasted rats (i.e., only water was available during a 12-h fast before and a 24-h fast after gentamicin injection) were used. The animals were synchronized on a 14-h light, 10-h dark cycle (lights on at 0600 h) for 1 week before gentamicin administration. In July 1993, each group of animals was treated with a single intraperitoneal injection of saline (NaCl, 0.9%) or gentamicin (150 mg/kg of body weight) at either the peak (1400 h) or the trough (0200 h) of the previously determined toxicity. On day 1, the 24-h urinary excretion of beta-galactosidase, N-acetyl-beta-D-glucosaminidase, and gamma-glutamyltransferase was significantly higher in normally fed animals treated with gentamicin at 1400 h than in their time-matched controls and in normally fed animals treated at 0200 h (P < 0.01), which had normal levels of these enzymes. By contrast, the urinary excretion of these enzymes was significantly higher in both groups of gentamicin-treated, fasted rats than in their time-matched control groups (P < 0.01), reaching levels similar to those measured in normally fed rats treated at 1400 h. The accumulation of gentamicin was significantly lower in the renal cortex of normally fed rats treated at 0200 h than in rats treated at 1400 h (P < 0.05), but this time-dependent difference was not found in fasted rats treated at 0200 and 1400 h. Immunogold labeling done on ultrathin sections and observed by electron microscopy showed a similar subcellular localization of gentamicin in normally fed and fasted rats treated at either 1400 or 0200 h. These results suggest that the feeding period is of crucial importance in the temporal variations of the nephrotoxicity of gentamicin in rats.
PMCID: PMC163178  PMID: 8851591
17.  Temporal variation in nephrotoxicity of low doses of isepamicin in rats. 
The temporal variation in the nephrotoxicity of low doses of isepamicin was studied in male Sprague-Dawley rats treated with a single daily intraperitoneal injection of saline (NaCl, 0.9%) or isepamicin (80 mg/kg of body weight) at either 0800, 1400, 2000, or 0200 h for 4 and 10 days. On day 10, the cellular regeneration (incorporation of [3H] thymidine into DNA of renal cortex) and cortical accumulation of isepamicin were significantly higher in animals treated at 1400 h than at 0200 h (P < 0.01). Immunogold labeling studies showed that isepamicin was essentially localized in the lysosomes of proximal tubular cells in all treated groups, but the density of the gold particles over the lysosomes was higher in animals treated at 1400 than at 0200 h. The results of the present study show that the renal toxicity of isepamicin was maximal at 1400 h (midlight period) and minimal at 0200 h (middark period).
PMCID: PMC163205  PMID: 8851618
18.  Comparative pharmacokinetics, distributions in tissue, and interactions with blood proteins of conventional and sterically stabilized liposomes containing 2',3'-dideoxyinosine. 
The pharmacokinetics and distribution in tissue of 2',3'-dideoxyinosine (ddI) encapsulated in sterically stabilized liposomes have been evaluated in rats. Most of the sterically stabilized liposomes concentrated in the spleen with a peak level at 24 h after their intravenous injection. An extended half-life in plasma was observed for sterically stabilized liposomes (14.5 h) compared with that of conventional liposomes (3.9 h). The systemic clearance of ddI incorporated in sterically stabilized liposomes was 180 times lower than that of the free drug. The levels of in vitro and in vivo protein binding on both conventional and sterically stabilized liposomes were also evaluated. Results suggest that the amount of proteins associated with liposomes might not be the only factor involved in the in vivo clearance of liposomes, as this process may also be influenced by the nature of the bound blood proteins.
PMCID: PMC163088  PMID: 8787911
19.  Differential increased survival of staphylococci and limited ultrastructural changes in the core of infected fibrin clots after daptomycin administration. 
A possible explanation for the difficulties encountered in curing deep fibrin-embedded infections is that antibiotic diffusion inside the infected fibrin matrix is not homogeneous and is insufficient to neutralize the pathogen. To evaluate this conjecture, the differential pharmacodynamics of daptomycin in fibrin clots infected with methicillin-susceptible and -resistant Staphylococcus aureus and Staphylococcus epidermidis was estimated. Daptomycin (20 or 50 mg/kg of body weight) was infused over 30 min. Fibrin clots and blood samples were evaluated from 0.5 to 42 h after the injections. The half-lives of daptomycin in serum and fibrin clot were close to identical after the two doses and averaged 5.4 and 22 h, respectively. The mean areas under the concentration-time curves from 0 to 42 h (AUC0-infinity) for daptomycin concentrations in serum and infected clots were 575 +/- 36.7 and 215 +/- 6.2 micrograms/g/h after administration of 20 mg/kg and 1,089 +/- 39.9 and 326 +/- 16.8 micrograms/g/h after administration of 50 mg/kg. A concentration gradient from the periphery to the core of the clots was observed in many clots up to 18 h after treatment. Mean peak concentrations in the core of the clots reached 60% of the peripheral values (P < 0.05) and were delayed for at least 3 h compared with the peripheral peak concentrations. AUC0-42 h of daptomycin concentration in the periphery and the core of clots were significantly different (P < 0.01). Survival of microorganisms was better in the core than in the periphery, with as much as a 3 log10 CFU/g difference between the center and the surface of the clot. Bacterial examination by transmission electron microscopy also showed noticeable differences in ultrastructural changes between those in the periphery and those in the core of the clots. In conclusion, the pharmacokinetics of daptomycin are significantly different at the periphery and within the core of fibrin clots, which may have led to the higher bacterial survival in the core of clots. Limited diffusion of daptomycin in fibrin, an essential component of the vegetation in bacterial endocarditis, could explain at least in part some of the treatment failures.
PMCID: PMC163083  PMID: 8787906
20.  Liposomal encapsulation of foscarnet protects against hypocalcemia induced by free foscarnet. 
Hypocalcemia and an increase in creatinine level are the most important serious effects associated with foscarnet (PFA) therapy. In an animal model, we have explored the potential protective role of liposome-encapsulated foscarnet (LE-PFA) on these metabolic abnormalities. PFA administered as one bolus injection (0.5 or 1.0 g/kg) caused significant rapid decreases (approximately 20%) in the levels of calcium and phosphorus in serum within a few minutes and up to 30 min after injection. LE-PFA did not induce any of these changes, while peak levels in serum and the half-life of this formulation were much higher than those of the free drug. PFA administered for 2 weeks (340 or 500 mg/kg/day) resulted in no changes in creatinine or blood urea nitrogen levels in serum at the low-dosage level, but at the higher-dosage level, the creatinine level in serum increased by day 5 posttreatment. Furthermore, there was no increase in the creatinine or blood urea nitrogen level after 2 weeks of treatment with LE-PFA at a dosage of 35 mg/kg/day. When the pharmacokinetics of both free PFA and LE-PFA were compared, the plasma half-life of the encapsulated drug was approximately four times longer than that of the free drug. In addition, the systemic clearance of LE-PFA was approximately one-fifth of that of the free drug. In conclusion, free PFA causes hypocalcemia and hypophosphatemia and increases the creatinine level in serum, whereas the LE form of this drug seems to protect against the abnormal changes in calcium and phosphorus levels caused by the free drug. By preventing hypocalcemia and increasing its half-life, LE-PFA can be used at lower doses and at longer intervals. Clinical investigations of these formulations may be worthwhile.
PMCID: PMC162866  PMID: 8540701
21.  Prolonged endotoxemia enhances the renal injuries induced by gentamicin in rats. 
The aim of this study was to evaluate the role of chronic endotoxemia in the nephrotoxicity of gentamicin (GM). Saline or Escherichia coli lipopolysaccharide (LPS) was administered to conscious rats by continuous intravenous perfusion (1 mg/kg per day for 7 days) from a subcutaneously implanted osmotic pump. Twenty-four hours after surgery (day zero), treatment with saline or GM (15 mg/kg; intraperitoneally, twice a day) was started for 5 days. Levels of LPS in plasma measured by Limulus amoebocyte lysate activity decreased significantly from days 1 through 8. At days 5 and 8, the cortical concentrations of GM were higher in the LPS-perfused and GM-treated group (LPS plus GM) than they were in the saline-perfused and GM-treated group (saline plus GM) (P less than 0.05). Blood urea nitrogen and serum creatinine remained at normal levels throughout the experiment. A significant increase of cortical tubular cell regeneration was observed in the LPS plus GM animals as compared with regeneration observed in the other groups (saline plus saline, LPS plus saline, and saline plus GM), as measured by [3H]thymidine incorporation into DNA. Moreover, histopathological nephrotoxicity scores showed a synergistic toxic effect between LPS and GM. These results demonstrate that chronic perfusion of low doses of LPS potentiates the nephrotoxicity of GM.
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PMCID: PMC171712  PMID: 2360824
22.  Influence of endotoxin on the intracortical accumulation kinetics of gentamicin in rats. 
The mechanism by which endotoxin (lipopolysaccharide [LPS]) modifies the intrarenal distribution and the nephrotoxic potential of gentamicin is unknown. We studied the influence of LPS on the intracortical accumulation kinetics of gentamicin in rats infused intravenously for 6 h, during which time steady-state levels of the antibiotic in serum were achieved. We compared gentamicin accumulation rates (V) in normal rats and in rats receiving LPS (0.5 and 5 mg/kg) as levels in serum (S) varied from 0.5 to 130 micrograms/ml. The pharmacokinetic parameters of gentamicin were previously measured in the three groups of rats that were studied in order to reach and maintain in each rat the desired levels of antibiotic in serum during the 6 h of infusion. Two hours before the infusion of gentamicin, LPS was injected intravenously over a period of 15 min. In normal rats, the increase in S was associated with a nonlinear increase in V. The Michaelis-Menten kinetics, which was the best-fitting function, gave an apparent Vmax (maximal capacity of uptake) of 195.03 +/- 9.75 micrograms/g per h and an apparent Km (concentration in serum at Vmax/2, an index of affinity) of 34.91 +/- 4.45 micrograms/ml (linear transformation of the experimental data by the Hanes-Woolf plot: r = 0.93, n = 85). In the rats that received LPS, the increase in S was associated with a linear increase of V: for LPS at 0.5 mg/kg, V = 27.00 + 1.50 S (r = 0.94, n = 80); for LPS at 5 mg/kg, V = 22.72 + 1.48 S (r = 0.94, n = 75). We conclude that endotoxin modifies the accumulation kinetics of gentamicin in the kidney cortices of rats.
PMCID: PMC171646  PMID: 2344164
23.  Influence of indomethacin on the intrarenal uptake of gentamicin in endotoxemic rats. 
Gentamicin is a commonly used antibiotic for the treatment of gram-negative-bacterial infections. Bacterial endotoxin is liberated during antibiotic therapy, and we have shown that endotoxemic animals accumulate more aminoglycosides in their renal parenchyma than normal animals. Vasoactive mediators, such as prostaglandins and thromboxanes, are released after endotoxin and are involved in inflammation. Indomethacin, a nonsteroidal anti-inflammatory drug known to inhibit the synthesis of these hormones, was infused intravenously as a bolus (3.0 mg/kg) or as a bolus followed by a continuous infusion (0.75 mg/kg per h) to rats given gentamicin. Levels of gentamicin in serum and kidney were increased 2 h post-antibiotic treatment in the endotoxemic animals. Renal function was not significantly disturbed. Indomethacin given as a bolus failed to correct the disturbed intrarenal pharmacokinetics of gentamicin induced by endotoxin. However, a bolus followed by continuous infusion of indomethacin resulted in low cortical and high papillary levels of antibiotic. These changes were correlated with the inhibition of prostaglandin synthesis from the kidney. These observations suggest an important role for prostaglandins in the interaction among endotoxin, aminoglycosides, and the kidney. Specific inhibitors of arachidonic acid metabolites should be investigated to further understand the mechanisms of this interaction.
PMCID: PMC172651  PMID: 2802560
24.  Isolation and biochemical characterization of Haemophilus species isolated simultaneously from the oropharyngeal and anogenital areas. 
Journal of Clinical Microbiology  1989;27(7):1486-1489.
Several reports have described the high frequency of pharyngeal isolation of Haemophilus species. Few studies have compared the simultaneous isolation rate of this species in the oropharyngeal and anogenital areas. Using two selective media, heart infusion agar (HIA) supplemented with 5% defibrinated rabbit blood, 1% IsoVitaleX, and either bacitracin alone (100 micrograms/ml) or bacitracin (5 micrograms/ml), vancomycin (3 micrograms/ml), and polymyxin B (1 microgram/ml), we isolated Haemophilus species in both areas in 89 of 399 (22.2%) patients consulting a sexually transmitted disease clinic. Of those, 56 were males and 33 were females. We recovered Haemophilus species in the oropharyngeal area in 384 patients (96%), while rectal and genital areas were colonized in 48 (12.0%) and 55 (13.8%) patients, respectively (both areas were colonized in 14 patients). Haemophilus parainfluenzae was isolated almost twice as often in the anogenital area as was H. influenzae. H. influenzae biotypes II and III and H. haemolyticus were the more prevalent XV-requiring haemophili isolated from the oropharynx, while H. influenzae biotype IV was more prevalent in the anogenital area. H. parainfluenzae biotypes I, II, and III were more prevalent in the oropharynx, while biotypes I and II were more prevalent in the anogenital area.
PMCID: PMC267600  PMID: 2671014
25.  Pharmacokinetics of carumonam after single and multiple 1- and 2-g dosage regimens. 
The pharmacokinetics of carumonam after single and multiple intravenous administration of 1- and 2-g dosage regimens were studied in 12 young male volunteers. Plasma and urine samples were collected in serial order for 24 h and assayed by high-pressure liquid chromatography. The mean elimination half-life of carumonam was not significantly affected by either dosage regimen or single dose versus steady state, ranging from 1.3 to 1.5 h. Mean concentrations at the end of the interval were not influenced by a multiple-dose administration. The normalized volume of distribution was independent of the dose, with values ranging from 0.16 to 0.19 liters/kg. After multiple administration, carumonam was cleared from the body more rapidly: from 96.2 to 121.7 ml/min after 1 g every 8 h, and from 102.1 to 122.3 ml/min after 2 g every 8 h (P less than 0.05). After 24 h, 75.0 to 80.7% of the dose was excreted unchanged in the urine. The protein binding of carumonam to human plasma remained stable at 28%. Carumonam was well tolerated by the volunteers.
PMCID: PMC172175  PMID: 3364953

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