Dosing regimen of gentamicin in neonates: evaluation of current guidelines based on a large population pharmacokinetic analysis

Objectives: Gentamicin is among the most commonly prescribed antibiotics in newborns, but large interindividual variability in exposure levels exists. Based on a population pharmacokinetic analysis of a cohort of unselected neonates, we aimed to validate current dosing recommendations from a recent reference guideline (Neofax®). Methods: From 3039 concentrations collected in 994 preterm (median gestational age 32.3 weeks, range 24.2-36.5) and 455 term newborns, treated at the University Hospital of Lausanne between 2006 and 2011, a population pharmacokinetic analysis was performed with NONMEM®. Model-based simulations were used to assess the ability of dosing regimens to bring concentrations into targets: trough ≤ 1mg/L and peak ~ 8mg/L. Results: A two-compartment model best characterized gentamicin pharmacokinetics. Model parameters are presented in the table. Body weight, gestational age and postnatal age positively influence clearance, which decreases under dopamine administration. Body weight and gestational age influence the distribution volume. Model based simulations confirm that preterm infants need doses superior to 4 mg/kg, and extended dosage intervals, up to 48 hours for very preterm newborns, whereas most term newborns would achieve adequate exposure under 4 mg/kg q. 24 h. More than 90% of neonates would achieve trough concentrations below 2 mg/L and peaks above 6 mg/L following most recent guidelines. Conclusions: Simulated gentamicin exposure demonstrates good accordance with recent dosing recommendations for target concentration achievement.

Photodynamic induced uptake of liposomal doxorubicin to rat lung tumors parallels tumor vascular density.

BACKGROUND: Visudyne®-mediated photodynamic therapy (PDT) at low drug/light conditions has shown to selectively enhance the uptake of liposomal doxorubicin in subpleural localized sarcoma tumors grown on rodent lungs without causing morphological alterations of the lung. The present experiments explore the impact of low-dose PDT on liposomal doxorubicin (Liporubicin™) uptake to different tumor types grown on rodent lungs. MATERIAL AND METHODS: Three groups of Fischer rats underwent subpleural generation of sarcoma, mesothelioma, or adenocarcinoma tumors on the left lung. At least five animals of each group (sarcoma, n = 5; mesothelioma, n = 7; adenocarcinoma, n = 5) underwent intraoperative low-dose (10 J/cm(2) at 35 mW/cm(2) ) PDT with 0.0625 mg/kg Visudyne® of the tumor and the lower lobe. This was followed by intravenous (IV) administration of 400 µg Liporubicin™. After a circulation time of 60 min, the tumor-bearing lung was processed for HPLC analyses. At least five animals per group underwent the same procedure but without PDT (sarcoma, n = 5; mesothelioma, n = 5; adenocarcinoma, n = 6). Five untreated animals per group underwent CD31 immunostaining of their tumors with histomorphometrical assessment of the tumor vascularization. RESULTS: Low-dose PDT significantly enhanced Liporubicin™ uptake to all tumor types (sarcoma, P = 0.0007; mesothelioma, P = 0.001; adenocarcinoma, P = 0.02) but not to normal lung tissue compared to IV drug administration alone. PDT led to a significantly increased ratio of tumor to lung tissue drug uptake for all three tumor types (P < 0.05). However, the tumor drug uptake varied between tumor types and paralleled tumor vascular density. The vascular density was significantly higher in sarcoma than in adenocarcinoma (P < 0.001) and mesothelioma (P < 0.001), whereas there was no significant difference between adenocarcinoma and mesothelioma. CONCLUSION: Low-dose Visudyne®-mediated PDT selectively enhances the uptake of systemically administered liposomal doxorubicin in tumors without affecting the drug uptake to normal lung. However, drug uptake varied significantly between tumor types and paralleled tumor vascular density.

Application of ZnO-Functionalised-Sepiolite in weaning piglet diets

The objective was to evaluate the effect of ZnO-Functionalised-Sepiolite (ZnO-Sepiolite) to fulfil Zn requirements and health status of weaning piglets. Pre-starter Basal Diet (BD, corn– soybean based, from weaning till 14 days on trial) was calculated to provide 27 mg Zn/kg feed from raw materials and had no added ZnO and no antibiotics or organic acids. Treatments during pre-starter period were: 1) BD+90% of NRC Zn requirements completed with ZnO (ZnO90); 2) BD+90% of NRC Zn requirements completed with ZnO-Sepiolite (ZnOS90); 3) BD+3000 mg ZnO/kg of diet (ZnO3000); 4) BD+150 mg added Zn/kg diet from ZnO-Sepiolite (ZnOS150). The starter feed (corn–soybean based, from 14 till 31 days on trial) was common for all piglets, and met 90% NRC Zn requirements by adding ZnO. Diarrhea affected more than 50% of the animals of ZnO90, ZnOS90 and ZnOS150, and 33% of the ZnO3000 animals. Animals from ZnOS90 tended (Pb0.10) to improve Gain to Feed ratio (G:F) compared to animals from ZnO90 (0.830 kg/kg vs. 0.811 kg/kg for G:F). Performance of animals from ZnO3000 was not significantly different from the other treatments, and was numerically similar to animals from ZnOS90. The inclusion of ZnO at 3000 mg/kg of feed in the pre-starter period numerically decreased P in serum at the end of this period, with no effect on Ca level; normal levels were restored after 2 weeks of feeding the same levels of Zn than other animals. Animals fed ZnOSepiolite diets had numerically higher serum Ca than ZnO90 and ZnO3000 at 12 days and higher than ZnO90 at 28 days. Serum Zn levels were significantly higher for ZnO3000 than the other treatments.

Strategies of use of a specific immunoglobulin-rich egg yolk powder in weaning piglets

Two experiments were performed to determine the best strategy of use of the product TRACTcare® 4P (ITPSA) (TC, specific immunoglobulin-rich egg yolk powder within an energetic fatty acid matrix) in piglets from weaning and for 6 weeks, in diets without or with inclusion of antibiotics. Each trial was performed with 144 piglets in 24 pens, in a completely randomized design blocked by initial body weight. Feeds were formulated according to animal requirements in two periods. In the first trial no antibiotics were included in the feeds and no room disinfection from previous trial was performed; treatments were: 1) Negative control (NC); 2) NC+TC on top of the feed within the hopper for the first 3 days on trial (30 g/pig×day), and eventually if diarrhea appeared (TCOT); 3) NC+TC ad libitum provided in an extra hopper within the pen (TCAL); and 4) NC+TC at 5 g/kg added to the feed in the mixer (TC5). In the second trial, treatments were: 1) Positive control: basal diet that included 250 mg/kg amoxiciline (BD)+100 mg/kg colistine (AC); 2) BD+2 g/kg TC (TC2A); 3) BD+5 g/kg TC (TC5A); and 4) BD+8 g/kg TC (TC8A). In diets without antibiotics, the product TC at 5 g/kg in the feed numerically improved BW by 8% compared to Control animals, while G:F was almost identical between both groups. When antibiotics were used in the feed, replacement of colistin at 100 mg/kg for TC at 2 g/kg in feed numerically improved the performance compared to Positive control animals (for the whole trial period ADG 8% better: 390 g vs. 361 g; G:F 1% better: 0.748 kg/kg vs. 0.742 kg/kg), possibly due to the stimulation of feed consumption at weaning. In both trials, the lower number of dead and culled animals from TC5 and TC2A together with higher BW represented an advantage over Control treatments of 6% to 10% animals more and 15% to 17% total BW more at the end of the trial.

Delayed otogenic hydrocephalus after acute otitis media in pediatric patients: the changing presentation of a serious otologic complication.

CONCLUSIONS: The clinical presentation of otogenic dural sinus thrombosis (DST) as a complication of acute otitis media (AOM) can be masked by antibiotic treatment. Morning episodes of vomiting and/or headache, visual impairment and a history of AOM seem to be indicative of otogenic hydrocephalus. We therefore advocate that the MRI scans of patients with similar symptoms should be carefully studied to facilitate the early diagnosis of a potentially life-threatening complication. OBJECTIVE: To describe the frequency, pathognomonic signs, clinical course and outcome of otogenic hydrocephalus and DST as complications of AOM in pediatric patients. MATERIAL AND METHODS: We undertook a retrospective chart review of all pediatric patients (age 1-14 years) treated for otitis media and its complications at an academic medical center between 1999 and 2003. The main outcome measures were otologic and ophthalmologic findings and CT and MRI scans at the beginning of treatment and 3 months later. RESULTS: We report on five cases with otogenic DST following AOM. All but one of them presented initially with diplopia caused by otogenic hydrocephalus. In four cases the otologic complaints had already disappeared by the time of MRI confirmation of the diagnosis. Only one child was referred with severe otologic symptoms. Management included systemic antibiotics, short-term heparin anticoagulation and surgical decompression. In our cases, even after intensive i.v. antibiotic treatment, only surgery led to a significant improvement in the clinical condition.

Medicinal products: regulation of biosynthesis in space and time

We live in a "Demon-Haunted World". Human health care requires the ever increasing resistance of pathogens to be confronted by a correspondingly fast rate of discovery of novel antibiotics. One of the possible strategies towards this objective involves the rational localization of bioactive phytochemicals. The conceptual basis of the method consists in the surprisingly little known gearings of natural products with morphology, ecology and evolution of their plant source, i. e. an introspection into the general mechanisms of nature.

A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria.

Bacteria can be refractory to antibiotics due to a sub-population of dormant cells, called persisters that are highly tolerant to antibiotic exposure. The low frequency and transience of the antibiotic tolerant "persister" trait has complicated elucidation of the mechanism that controls antibiotic tolerance. In this study, we show that 2' Amino-acetophenone (2-AA), a poorly studied but diagnostically important small, volatile molecule produced by the recalcitrant gram-negative human pathogen Pseudomonas aeruginosa, promotes antibiotic tolerance in response to quorum-sensing (QS) signaling. Our results show that 2-AA mediated persister cell accumulation occurs via alteration of the expression of genes involved in the translational capacity of the cell, including almost all ribosomal protein genes and other translation-related factors. That 2-AA promotes persisters formation also in other emerging multi-drug resistant pathogens, including the non 2-AA producer Acinetobacter baumannii implies that 2-AA may play an important role in the ability of gram-negative bacteria to tolerate antibiotic treatments in polymicrobial infections. Given that the synthesis, excretion and uptake of QS small molecules is a common hallmark of prokaryotes, together with the fact that the translational machinery is highly conserved, we posit that modulation of the translational capacity of the cell via QS molecules, may be a general, widely distributed mechanism that promotes antibiotic tolerance among prokaryotes.

Multiple isolates from Aids patients: aspects of an analysis by a genotypic marker and antimicrobial susceptibilities variations

Twenty-one Mycobacterium avium multisolates, from ten human immunodeficiency virus-infected patients, were typed by restriction fragment length polymorphism using as marker the IS1245 and characterized by minimum inhibitory concentration for nine different antibiotics. Two out of four patients harboring multisolates with different fingerprint profile, were therefore considered as having a polyclonal infection, since their isolates were taken from sterile site. This result confirms that polyclonal infection caused by M. avium occurs with a nonnegligenciable frequency. Analyzing the multisolates susceptibility profile of each patient it was observed that most of them were infected with strains having appreciably different antimicrobial susceptibility patterns, no matter what the genotypic pattern of the strains was. These results have strong implication for the treatment of the patients.

Prevalence, species differentiation, haemolytic activity, and antibiotic susceptibility of aeromonads in untreated well water

The use of untreated water for drinking and other activities have been associated with intestinal and extraintestinal infections in humans due to Aeromonas species. In the present study aeromonads were isolated from 48.7% of 1,000 water samples obtained from wells and other miscellaneous sources. Aeromonas species were detected in 45% of samples tested in spring, 34.5% in summer, 48% in autumn and 60% of samples tested in winter. Speciation of 382 strains resulted in 225 (59%) being A. hydrophila, 103 (27%) A. caviae, 42 (11%) A. sobria and 11 (3%) atypical aeromonads. Of 171 Aeromonas strains tested for their haemolytic activity, 53%, 49%, 40% and 37% were positive in this assay using human, horse, sheep and camel erythrocytes respectively. The results obtained indicate that potentially enteropathogenic Aeromonas species are commonly present in untreated drinking water obtained from wells in Libya (this may also apply to other neighbouring countries) which may pose a health problem to users of such water supplies. In addition, ceftriaxone and ciprofloxacin are suitable drugs that can be used in the treatment of Aeromonas-associated infections, particularly in the immunocompromised, resulting from contact with untreated sources of water.

Microbiological diagnosis of community-acquired respiratory tract infections by nucleic acid detection.

Background: Microbiological diagnostic procedures have changed significantly over the last decade. Initially the implementation of the polymerase chain reaction (PCR) resulted in improved detection tests for microbes that were difficult or even impossible to detect by conventional methods such as culture and serology, especially in community-acquired respiratory tract infections (CA-RTI). A further improvement was the development of real-time PCR, which allows end point detection and quantification, and many diagnostic laboratories have now implemented this powerful method. Objective: At present, new performant and convenient molecular tests have emerged targeting in parallel many viruses and bacteria responsible for lower and/or upper respiratory tract infections. The range of test formats and microbial agents detected is evolving very quickly and the added value of these new tests needs to be studied in terms of better use of antibiotics, better patient management, duration of hospitalization and overall costs. Conclusions: Molecular tools for a better microbial documentation of CA-RTI are now available. Controlled studies are now required to address the relevance issue of these new methods, such as, for example, the role of some newly detected respiratory viruses or of the microbial DNA load in a particular patient at a particular time. The future challenge for molecular diagnosis will be to become easy to handle, highly efficient and cost-effective, delivering rapid results with a direct impact on clinical management.

Y-688, a new quinolone active against quinolone-resistant Staphylococcus aureus: lack of in vivo efficacy in experimental endocarditis.

Y-688 is a new fluoroquinolone with increased activity against ciprofloxacin-resistant staphylococci. The MICs of Y-688 and other quinolones were determined for 58 isolates of ciprofloxacin-resistant and methicillin-resistant Staphylococcus aureus (MRSA). The MICs at which 50% and 90% of bacteria were inhibited were &gt;/=128 and &gt;/=128 mg/liter, respectively, for ciprofloxacin, 16 and 32 mg/liter, respectively, for sparfloxacin, and 0.25 and 1 mg/liter, respectively, for Y-688. This new quinolone was further tested in rats with experimental endocarditis due to either of two isolates of ciprofloxacin-resistant MRSA (namely, P8/128 and CR1). Infected animals were treated for 3 days with ciprofloxacin, vancomycin, or Y-688. Antibiotics were administered through a computerized pump to simulate human-like pharmacokinetics in the serum of rats. The anticipated peak and trough levels of Y-688 were 4 and 1 mg/liter at 0.5 and 12 h, respectively. Treatment with ciprofloxacin was ineffective. Vancomycin significantly decreased vegetation bacterial counts for both organisms (P less, similar 0.05). In contrast, Y-688 only marginally decreased vegetation bacterial counts (P greater, similar 0.05). Moreover, several vegetation that failed Y-688 treatment grew staphylococci for which the MICs of the test antibiotic were increased two to eight times. Y-688 also selected for resistance in vitro, and isolates for which the MICs were increased eight times emerged at a frequency of ca. 10(-8). Thus, in spite of its low MIC for ciprofloxacin-resistant MRSA, Y-688 failed in vivo and its use carried the risk of resistance selection. The fact that ciprofloxacin-resistant staphylococci became rapidly resistant to this potent new drug suggests that the treatment of ciprofloxacin-resistant MRSA with new quinolones might be more problematic than expected.

Deletion of hypothetical wall teichoic acid ligases in Staphylococcus aureus activates the cell wall stress response.

The Staphylococcus aureus cell wall stress stimulon (CWSS) is activated by cell envelope-targeting antibiotics or depletion of essential cell wall biosynthesis enzymes. The functionally uncharacterized S. aureus LytR-CpsA-Psr (LCP) proteins, MsrR, SA0908 and SA2103, all belong to the CWSS. Although not essential, deletion of all three LCP proteins severely impairs cell division. We show here that VraSR-dependent CWSS expression was up to 250-fold higher in single, double and triple LCP mutants than in wild type S. aureus in the absence of external stress. The LCP triple mutant was virtually depleted of wall teichoic acids (WTA), which could be restored to different degrees by any of the single LCP proteins. Subinhibitory concentrations of tunicamycin, which inhibits the first WTA synthesis enzyme TarO (TagO), could partially complement the severe growth defect of the LCP triple mutant. Both of the latter findings support a role for S. aureus LCP proteins in late WTA synthesis, as in Bacillus subtilis where LCP proteins were recently proposed to transfer WTA from lipid carriers to the cell wall peptidoglycan. Intrinsic activation of the CWSS upon LCP deletion and the fact that LCP proteins were essential for WTA-loading of the cell wall, highlight their important role(s) in S. aureus cell envelope biogenesis.

Drug resistance and genotypes of strains of Mycobacterium tuberculosis isolated from human immunodeficiency virus-infected and non-infected tuberculosis patients in Bauru, São Paulo, Brazil

Little is known about transmission and drug resistance of tuberculosis (TB) in Bauru, State of São Paulo. The objective of this study was to evaluate risk factors for transmission of Mycobacterium tuberculosis strains in this area. Strains were collected from patients attended at ambulatory services in the region and susceptibility towards the main first line antibiotics was determined and fingerprinting performed. A total of 57 strains were submitted to susceptibility testing: 23 (42.6%) were resistant to at least one drug while 3 (13%) were resistant against both rifampicin and isoniazide. Resistant strains had been isolated from patients that had not (n = 13) or had (n = 9) previously been submitted to anti-TB treatment, demonstrating a preoccupying high level of primary resistance in the context of the study. All strains were submitted to IS6110 restriction fragment length polymorphism (IS6110-RFLP) and double repetitive element PCR (DRE-PCR). Using IS6110-RFLP, 26.3% of the strains were clustered and one cluster of 3 patients included 2 HIV-infected individuals that had been hospitalized together during 16 days; clustering of strains of patients from the hospital was however not higher than that of patients attended at health posts. According to DRE-PCR, 55.3% belonged to a cluster, confirming the larger discriminatory power of IS6110-RFLP when compared to DRE-PCR, that should therefore be used as a screening procedure only. No clinical, epidemiological or microbiological characteristics were associated with clustering so risk factors for transmission of TB could not be defined in the present study.

The effect of nitric oxide combined with fluoroquinolones against Salmonella enterica serovar Typhimurium in vitro

Two regulons, soxRS and marRAB, are associated with resistance to quinolones or multiple antibiotic in Salmonella enterica serovar Typhimurium. These regulons are activated by nitric oxide and redox-cycling drugs, such as paraquat and cause on activation of the acrAB-encoded efflux pump. In this study, we investigated the effect of nitric oxide (NO) alone and in combination with ofloxacin, ciprofloxacin, and pefloxacin against S. typhimurium clinical isolates and mutant strains in vitro. We did not observe synergistic effect against clinical isolates and SH5014 (parent strain of acr mutant), while we found synergistic effect against PP120 (soxRS mutant) and SH7616 (an acr mutant) S. typhimurium for all quinolones. Our results suggest that the efficiencies of some antibiotics, including ofloxacin, ciprofloxacin, and pefloxacin are decreased via activation of soxRS and marRAB regulons by NO in S. enterica serovar Typhimurium. Further studies are warranted to establish the interaction of NO with the genes of Salmonella and, with multiple antibiotic resistance.

Characterization of [Ca2+]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes

Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1% while A23187 (0.5 µM) alone did not induce significant effects (17%); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC.