Antibiotic efflux pumps in prokaryotic cells: occurrence, impact on resistance and strategies for the future of antimicrobial therapy.
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Animals↗
Biomedical subjects
J C Pechère
Publications and source records attributed to J C Pechère.
Influence of previous exposure to antibiotic therapy on the susceptibility pattern of Pseudomonas aeruginosa bacteremic isolates.
Many patients who present with Pseudomonas aeruginosa bacteremia have been previously exposed to antibiotics. To assess whether resistance of bacteremic strains to antipseudomonal antibiotics (piperacillin, ceftazidime, imipenem, ciprofloxacin, or aminoglycosides) is associated with previous exposure to these drugs, a case-control study including 267 cases of P. aeruginosa bacteremia was conducted. Twenty-five percent of the episodes had been preceded by the exposure to an antipseudomonal antibiotic. Eighty-one strains were resistant to at least 1 antibiotic; 186 were susceptible to all drugs. Via univariate analysis, the risks of resistance to ceftazidime and imipenem were found to be significantly associated with previous receipt of these agents. Using multivariate analysis, exposure to any antipseudomonal antibiotic as a monotherapy was found to be associated with an increased risk of subsequent resistance to itself (odds ratio, 2.5; P=.006). Therefore, clinicians should avoid readministering previously prescribed antibiotics when initiating empiric therapies for possible P. aeruginosa bacteremia, especially when they have been given as monotherapies.
Adolescent↗
Patients' interviews and misuse of antibiotics.
To better evaluate patient contribution in antibiotic use, we questioned 5379 subjects from 9 countries. Antibiotics are perceived as strong, efficient drugs, but they are believed to undermine immunity. Interviewees believe that most respiratory infections, except the common cold, require antibiotic therapy, and 11% of them had to exaggerate their symptoms to get an antibiotic prescription from their physician. About 1 patient in 4 saved part of the antibiotic course for future use. Sixty-nine percent of the patients claimed to have taken the course until the end (United Kingdom, 90%; Thailand, 53%), and 75% claimed that they actually took all the daily doses. In all countries, it was possible to get antibiotics from a pharmacist without a medical prescription. This study shows that patients exert pressure on their doctors to get antibiotics and should allow a design for precise educational action aimed at the public for better control of antibiotic use in the community.
Adult↗
Overexpression, refolding, and purification of the histidine-tagged outer membrane efflux protein OprM of Pseudomonas aeruginosa.
This paper describes the overproduction and purification of the C-terminus polyhistidine-tagged outer membrane protein OprM, which is a part of the MexA-MexB-OprM active efflux system of Pseudomonas aeruginosa. Renaturation of the protein from inclusion bodies of Escherichia coli was achieved using guanidine-HCl as denaturing agent and n-octylpolyoxyethylene (C8POE) and n-octyltetraoxyethylene (C8E4) as nonionic detergents. The refolded protein was purified by ion-exchange and nickel-affinity chromatography. The final yield was 6 mg of pure histidine-tagged OprM per liter of E. coli culture. Renaturation was monitored by the effects of heating prior to SDS-PAGE, using a typical and exclusive property of outer membrane proteins. Immunoblotting revealed that the recombinant protein is addressed to the outer membrane of E. coli, after maturation by excision of its N-terminal signal sequence. Complementation of an oprM deletion mutant with the plasmid encoded histidine-tagged OprM protein restored antibiotic susceptibilities to wild-type levels, demonstrating functionality of recombinant OprM.
Affinity Labels↗
Experimental treatment of vascular graft infection due to Staphylococcus epidermidis by in situ replacement with a rifampin-bonded polyester graft.
In situ prosthetic graft replacement (ISPGR) of an infected prosthesis raises the risk of recurrent infection in the new graft, especially in cases involving drug-resistant microorganisms. The purpose of this animal study was to evaluate in situ replacement of a vascular graft infected by a highly rifampin-resistant strain of Staphylococcus epidermidis with the use of a rifampin-bonded polyester graft. Antibiotic bonding was obtained by soaking grafts in a high dose of rifampin solution (60 mg/mL). The infrarenal abdominal aorta of 20 dogs was replaced using a polyester prosthesis infected with a highly rifampin-resistant strain of Staphylococcus epidermidis. One week later, the 18 surviving animals were randomized into three groups. Group I (n = 6) did not undergo reoperation. Group II (n = 6) underwent ISPGR using a rifampin-bonded prosthesis. Group III (n = 6) underwent ISPGR using an untreated prosthesis. All surviving animals were killed 28 days after the first procedure. Infectious signs were noted and bacteriological study was carried out on explanted prostheses and various tissue samples. The findings of this experimental study show that soaking a polyester prosthesis in a high-dose rifampin solution can prevent reinfection after in situ replacement of a prosthesis infected by a highly rifampin-resistant Staphylococcus epidermidis.
Anastomosis, Surgical↗
New perspectives on macrolide antibiotics.
Macrolides are not used exclusively for the treatment of community-acquired respiratory tract infections. Their ability to penetrate cells makes them highly suitable for the treatment of diseases caused by intracellular pathogens, such as non-gonococcal urethritis and trachoma. Azithromycin is approved for these indications. Clinical studies have also been conducted, or are currently being carried out, to assess the use of macrolides in the treatment of atherosclerosis, eradication of Helicobacter pylori and the management of life-threatening gastrointestinal diseases, cystic fibrosis and malaria.
Anti-Bacterial Agents↗
Macrolide resistance mechanisms in Gram-positive cocci.
Two principal mechanisms of resistance to macrolides have been identified in Gram-positive bacteria. Erythromycin-resistant methylase is encoded by erm genes. Resultant structural changes to rRNA prevent macrolide binding and allow synthesis of bacterial proteins to continue. Presence of the erm gene results in high-level resistance. Modification of the mechanism whereby antibiotics are eliminated from the bacteria also brings about resistance. Bacteria carrying the gene encoding macrolide efflux (i.e. the mefE gene) display relatively low-level resistance. Azithromycin, because of its ability to achieve concentrations at sites of infections, is capable of eradicating mefE-carrying strains. Other resistance mechanisms, involving stimulation of enzymatic degradation, appear not to be clinically significant.
Anti-Bacterial Agents↗
C-terminal region of Pseudomonas aeruginosa outer membrane porin OprD modulates susceptibility to meropenem.
We investigated the unusual susceptibility to meropenem observed for seven imipenem-resistant clinical isolates of Pseudomonas aeruginosa. These strains were genetically closely related, expressed OprD, as determined by Western blot analyses, and were resistant to imipenem (>5 microg/ml) but susceptible to meropenem (<1 microg/ml). The oprD genes from two isolates were entirely sequenced, and their deduced protein sequences showed 93% identity with that of OprD of strain PAO1. The major alteration consisted of the replacement of a stretch of 12 amino acids, located in putative external loop L7 of OprD, by a divergent sequence of 10 amino acid residues. The oprD gene variants and the wild-type oprD gene were cloned and expressed in a defined oprD mutant. The meropenem MICs for strains carrying the oprD genes from clinical isolates were four times lower than that for the strain carrying the wild-type oprD gene. Imipenem activities, however, were comparable for all strains. Furthermore, meropenem hypersusceptibility was obtained with a hybrid OprD porin that consisted of the PAO1 oprD gene containing loop L7 from a clinical isolate. These results show that the C-terminal portion of OprD, in particular, loop L7, was responsible for the unusual meropenem hypersusceptibility. Competition experiments suggested that the observed OprD modifications in the clinical isolates did not affect antagonism between imipenem and the basic amino acid L-lysine. We further propose that shortening of putative loop L7 of the OprD porin by 2 amino acid residues sufficiently opens the porin channel to allow optimal penetration of meropenem and increase its activity. In contrast, this alteration would not affect susceptibility to a smaller carbapenem molecule, such as imipenem.
Amino Acid Sequence↗
Genetic background of attenuated Salmonella typhimurium has profound influence on infection and cytokine patterns in human dendritic cells.
Salmonella typhimurium (ST) can cause infection in man, and attenuated strains are under consideration as live vaccine vectors. However, little is known about the interaction of ST with human dendritic cells (DC). Here, we compared the consequences of exposure of human, monocyte-derived DC with different attenuated strains of ST. Infection was observed with all four strains tested (wild type, PhoP-, PhoPc, and AroA), but the PhoPc strain was by far the most efficient. Intracellular persistence of wild type and PhoP- was longer than that of PhoPc and AroA, both of which were largely eliminated within 24 h. Most DC survived infection by the attenuated strains, although apoptosis was observed in a fraction of the exposed cells. All strains induced DC maturation, independent from the extent of infection. Although all strains stimulated secretion of TNF-alpha and IL-12 strongly, PhoPc induced significantly less IL-10 than the other three strains and as much as 10 times less IL-10 than heat-killed PhoPc, suggesting that this mutant suppressed the secretion of IL-10 by the DC. These data indicate that infectivity, bacterial elimination, and cytokine secretion in human DC are controlled by the genetic background of ST.
3-Phosphoshikimate 1-Carboxyvinyltransferase↗
Azithromycin reduces the production of virulence factors in Pseudomonas aeruginosa by inhibiting quorum sensing.
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Anti-Bacterial Agents↗
New strategies against an old plague: genetically engineered tuberculosis vaccines.
The failure of BCG vaccination to control the global tuberculosis epidemic and the spread of multidrug resistance underline the need for a better vaccine. Recent advances in molecular microbiology, gene therapy, and, last but not least, immunobiology, provide a rational basis for the development of more efficient vaccines against tuberculosis. The complete sequencing of the M. tuberculosis genome marked a turning point in tuberculosis vaccine research. The advent of genetic vaccination with either naked DNA, live recombinant, or artificial vaccine vectors holds out great promise of more efficient immunisation, in particular against intracellular pathogens such as M. tuberculosis. Our new understanding of how the immune response is orchestrated by dendritic cells makes it possible to design vaccines which specifically exploit the functions of these antigen presenting cells. Yet M. tuberculosis has kept many of its secrets, and although functional genomics, molecular medicine and immunotherapy are evolving rapidly, much empiric search and discovery are needed until the "captain of all these men of death" gives up his ghost to biotechnology.
BCG Vaccine↗
"Chance makes a football of man's life".
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Humans↗
Optimizing economic outcomes in antibiotic therapy of patients with acute bacterial exacerbations of chronic bronchitis.
The social, medical and economic effects of acute bacterial exacerbations of chronic bronchitis on individual patients and the resource implications of this disease for the healthcare sector are considerable. Optimizing the selection of patients who should receive antibiotics according to stringent clinical criteria is the first step in promoting good clinical practice and cost-effectiveness. Antibiotic efficacy is then the major driver of cost, especially when it reduces the need for hospitalization. Resistance to first-line antibiotics can be expected to increase the risk of treatment failure. Other drivers of cost include non-compliance, which predisposes to therapeutic failure, and the selection of resistant strains. Treatment regimens of short duration, once-daily dosing and good tolerability are determinants of good compliance and cost savings. The expenses of first-line antibiotics typically account for only a small proportion of the overall costs of healthcare and the cheapest antibiotics are not necessarily the most cost-effective. The clinical success rate of first-line therapy is the primary determinant of the overall expenditure on healthcare because of the high costs associated with treatment failure, especially if it leads to hospitalization. Factors such as poor patient compliance and high antibiotic resistance rates, which undermine the clinical efficacies of first-line therapy, will increase the overall costs of treatment.
Acute Disease↗
Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili.
We describe swarming in Pseudomonas aeruginosa as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in P. aeruginosa is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a fliC mutant of P. aeruginosa displayed slightly diminished swarming, a pilR and a pilA mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the las cell-to-cell signaling system showed diminished swarming behavior, while rhl mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P. aeruginosa in this type of surface motility.
Aspartic Acid↗
Parameters important in short antibiotic courses.
The aim of antibiotic therapy is pathogen eradication, which is often assumed on the basis of the alleviation of the signs and symptoms of the disease. Pharmacokinetic/pharmacodynamic in vitro and animal models have now been developed to predict bacteriological efficacy and to establish dosing regimens that are effective and control the development of resistance. These models may be applied to the evaluation of new short-course dosing regimens, lasting no longer than 3 - 5 days. Single-dose therapy, using agents with a prolonged duration of action, is currently employed for streptococcal pharyngitis, uncomplicated gonorrhoea and uncomplicated lower urinary tract infections in women. The use of short-course therapy in immunocompetent patients for the treatment of community-acquired infections that have a low bacterial load is feasible. Such regimens may have a number of advantages over those currently employed, which typically involve dosing for 7 - 10 days. These advantages include improved tolerability, reduction in healthcare costs, enhanced patient compliance and the prevention of the emergence of resistance.
Animals↗
Bacterial antibiotic efflux systems of medical importance.
Multidrug efflux systems endow on bacterial cells the ability to limit the access of antimicrobial agents to their targets. By actively pumping out antibiotic molecules, these systems prevent the intracellular accumulation necessary for antibiotics to exert their lethal activity. Drug efflux appears to be one of the most widespread antibiotic resistance mechanisms among microorganisms, since it has been demonstrated to occur in many Gram-positive and Gram-negative bacteria including medically important species like staphylococci, streptococci, enterobacteria and opportunistic pathogens like Pseudomonas aeruginosa. Efflux pumps can be specific for only one substrate or accommodate a more or less wide range of noxious products. Export of structurally unrelated compounds confers a multidrug-resistance phenotype on bacterial cells. Therapeutically critical levels of resistance can be achieved by overexpression of efflux systems, especially in those species such as P. aeruginosa which possess a low outer membrane permeability. It is suspected that the dual physiological function of active efflux systems is both the secretion of intracellular metabolites and the protection against a variety of harmful substances that the microorganism may encounter in its natural environment.
Bacteria↗
Evaluation of trimethoprim and sulphamethoxazole as monotherapy or in combination in the management of toxoplasmosis in murine models.
The combination of trimethoprim (TMP) and sulphamethoxazole (SMX) is commonly used for the prevention of cerebral toxoplasmosis although there is no firm experimental basis to support this regimen. We used strain RH tachyzoites for challenge in the acute murine model of toxoplasmosis and found that TMP administered as a single agent, failed to eradicate toxoplasma even at the highest dose (70 mg/kg per day). SMX alone at 600 mg/kg per day, protected ten out of ten mice, although inoculation of brain from surviving animals to naive mice resulted in the development of an encephalitis. When combined, TMP (60 mg/kg per day) and SMX (300 mg/kg per day) protected ten out of ten mice and gave a 'cure' in four out of four mice. In the chronic cystogenic murine models, the combination TMP plus SMX administered from day 5 for 15 days or from day 28 for 288 days, gave protection and even apparent toxoplasmal eradication ('cure') at the highest dosing (60/300 mg/kg per day). However, microscopic severe encephalitis was found in mice classified as 'cured' after reinoculation. This result makes the interpretation of 'cure' very difficult. In conclusion TMP and SMX act synergistically, SMX being the more active arm of the combination. The combination was efficient in preventing the lethal development of chronic toxoplasma encephalitis, but did not guarantee complete recovery.
Animals↗