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P Plesiat

Publications and source records attributed to P Plesiat.

At least 19 recordsLinked to original sources
PubMed2006

In-vivo impact of the MexXY efflux system on aminoglycoside efficacy in an experimental model of Pseudomonas aeruginosa pneumonia treated with tobramycin.

Aminoglycosides are of major importance in treating Pseudomonas aeruginosa pneumonia (PAP). However, their efficacy may be compromised by low-level resistance caused by the inducible MexXY multidrug efflux pump. In the present study, the impact of the MexXY efflux pump was investigated in vivo in an experimental model of PAP in rabbits treated with intravenous tobramycin. Three strains were used to induce PAP in rabbits: PAO1 (wild-type strain; MIC 1 mg/L), mutant 11B (mexX::Tn501; no expression of MexXY; MIC 0.5 mg/L) and mutant MutGR1 (MexZ null; constitutive expression of MexXY; MIC 2 mg/L). Five hours after inoculation, treatment with tobramycin (10 mg/kg) was implemented (peak serum concentration 30 mg/L). The animals were killed humanely 48 h after inoculation, and the residual pulmonary bacterial concentration was determined. Selection of bacteria expressing MexXY was determined by plating lung homogenates on agar plates containing antibiotic. Mean bacterial counts (log(10) CFU/g) for treated vs. untreated rabbits were 6.26 and 8.13 (p < 0.0001), 6.00 and 8.38 (p < 0.001), and 7.25 and 8.79 (p 0.04) for PAO1, 11B and MutGR1, respectively, with an overall mortality rate of 0% vs. 8.9% (p < 0.01). MexXY-overexpressing bacteria were recovered from three (21%) treated rabbits. The C(max)/MIC ratio was the parameter that was best associated with tobramycin efficacy. The bacteria overexpressing MexXY, recovered from lung, occurred with a C(max)/MIC window of 19-26. It was concluded that the experimental PAP model highlights poor tobramycin bacteriological efficacy in vivo, contrasting with survival gain, and that the contribution of the MexXY system to this low level of tobramycin efficacy is modest. Finally, this model appears to be suitable for the investigation of new anti-pseudomonal therapeutic strategies.

Animals↗
PubMed2005

Susceptibility of Escherichia coli to the amoxycillin-clavulanate combination: which recommendations should be used to provide relevant information to clinicians?

This study compared MIC distributions of amoxycillin-clavulanate obtained with NCCLS and French (Comite de l'Antibiogramme de la Societe Francaise de Microbiologie; CA-SFM) methodologies for Escherichia coli isolates from urine that were non-susceptible to amoxycillin-clavulanate by the disk diffusion method. With the NCCLS and CA-SFM methods, 74% and 13%, respectively, of these isolates were susceptible to amoxycillin-clavulanate. Therefore, the apparent relatively poor efficacy of amoxycillin-clavulanate against E. coli in French hospitals probably reflects a methodological difference rather than a localised resistance problem. This implies that amoxycillin-clavulanate could be used as an alternative to fluoroquinolones for treatment of E. coli urinary tract infections. Susceptibility tests for amoxycillin-clavulanate should be standardised worldwide.

Amoxicillin-Potassium Clavulanate Combination↗
PubMed2003

Bacteremia caused by a novel species of Sphingobacterium.

A Gram-negative rod was isolated from the blood cultures of an 84-year-old man with foot cellulitis. The bacterium was first identified as Sphingobacterium spiritivorum on the basis of standard assimilation tests. However, sequencing analysis of its 16S rRNA genes and whole genome hybridization studies with other related bacteria showed that this isolate belongs to a so far undescribed species of Sphingobacterium, close to S. mizutae. This bacterium was susceptible to most of the antibiotics tested, including glycopeptides, but was resistant to aminoglycosides and polymyxins. Treatment with amoxicillin-clavulanate cured the infection.

Aged↗
PubMed2002

Mechanisms of beta-lactam resistance in Pseudomonas aeruginosa: prevalence of OprM-overproducing strains in a French multicentre study (1997).

One hundred and forty-three non-repetitive strains of Pseudomonas aeruginosa were collected in 13 French hospitals in 1997. A decreased susceptibility or resistance to ticarcillin (MIC > 16 mg/L) was found in 61 isolates (43%) and this was attributed to three major mechanisms: (i) overexpression of OprM and hence related efflux components such as MexAB or MexXY (42.6%), (ii) production of acquired beta-lactamase (29.5%) and (iii) overexpression of chromosomally encoded AmpC cephalosporinase (21.3%). Four of seven 'intrinsically' resistant strains (11.5%) with normal amounts of OprM were shown to produce low levels of AmpC, whereas in three isolates no resistance mechanism to beta-lactams could be identified. Overproduction of OprM thus appears as an important mechanism of ticarcillin resistance in French isolates of P. aeruginosa.

Bacterial Outer Membrane Proteins↗
PubMed2001

Cost-effectiveness of urinary dipsticks to screen asymptomatic catheter-associated urinary infections in an intensive care unit.

OBJECTIVE: To assess the cost-effectiveness of urinary dipsticks (UDs) to screen asymptomatic catheterized patients for quantitative urine. DESIGN: Prospective comparison of UD with quantitative urine culture (QUC) (reference technique) and cost-effectiveness analysis performed from the hospital's perspective. SETTING: Medical intensive care unit (ICU) of the Besançon University Hospital (France). PATIENTS AND PARTICIPANTS: All consecutive, asymptomatic, catheterized patients. INTERVENTIONS: Urinary dipsticks (Multistix 8-SG) were analyzed by the reflectance spectrophotometric method (Clinitek 50). Sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) of four combinations of the leukocyte (L) test pad and the nitrite (N) test pad were calculated: L and N, L or N, L alone and N alone. A micro-costing technique was used to determine the direct medical cost of each strategy. The calculated cost-effectiveness ratio was the incremental cost-effectiveness (ICE) ratio. MEASUREMENTS AND RESULTS: Three hundred thirty-nine urine samples taken from 144 patients were analyzed. The incidence of asymptomatic catheter-associated urinary tract infections (CAUTIs) was 31.3% (> or =10(5) organisms/ml). The L or N combination was the best detector of asymptomatic CAUTI: Se=87.2%, Sp=61.6%, PPV=30.6% and NPV=96.1%. The cost of QUC strategy and UD strategy was EUR 21.5 and EUR 12.6 per test, respectively. The ICE ratio of QUCs was EUR 69.5 per case of detected CAUTI. CONCLUSION: The UD is a cost-effective test for screening asymptomatic catheterized patients for quantitative urine culture in a medical ICU.

Cost-Benefit Analysis↗
PubMed2001

Effect of beta-lactam antibiotics on the in vitro development of resistance in Pseudomonas aeruginosa.

OBJECTIVE: To investigate whether stepwise selection of resistance mutations may mirror the continued bacterial exposure to antibiotics that occurs in the clinical setting. METHODS: We examined the in vitro development of resistance to a number of commonly used antibiotics (cefepime, cefpirome, ceftazidime, cefotaxime, piperacillin and imipenem) in Pseudomonas aeruginosa, a significant nosocomial pathogen. Stepwise resistance was assessed by serial passage of colonies located nearest to the inhibition zone on antibiotic-containing gradient plates. RESULTS: The lowest frequencies of spontaneous resistance mutations were found with cefepime and imipenem; these drugs also resulted in the slowest appearance of resistance of spontaneous resistance mutations. In five wild-type P. aeruginosa strains, cefepime-selected isolates required a mean of 30 passages to reach resistance; resistance occurred more rapidly in strains selected with other cephalosporins. P. aeruginosa strains that produced beta-lactamase or non-enzymatic resistance generally developed resistance more rapidly than wild-type strains. For most strains, resistance to all antibiotics except imipenem correlated with increased levels of beta-lactamase activity. Cross-resistance of cephalosporin-selected resistant mutants to other cephalosporins was common. Cephalosporin-resistant strains retained susceptibility to imipenem and ciprofloxacin. CONCLUSIONS: From our in vitro study, we can conclude that the rate of development of resistance of P. aeruginosa is lower with cefepime compared with other cephalosporines.

Anti-Bacterial Agents↗
PubMed1999

Propagation of TEM- and PSE-type beta-lactamases among amoxicillin-resistant Salmonella spp. isolated in France.

A survey conducted between 1987 and 1994 at the University Hospital of Besançon, France, demonstrated a dramatic increase (from 0 to 42. 5%) in the prevalence of amoxicillin resistance among Salmonella spp. Of the 96 resistant isolates collected during this period (including 77 Typhimurium), 54 were found to produce TEM-1 beta-lactamase, 40 produced PSE-1 (equivalent to CARB-2), one produced PSE-1 plus TEM-2, and one produced OXA-1 in isoelectric focusing and DNA hybridization experiments. Plasmids coding for these beta-lactamases were further characterized by (i) profile analysis, (ii) restriction fragmentation pattern analysis, (iii) hybridization with an spvCD-orfE virulence probe, and (iv) replicon typing. In addition, isolates of S. typhimurium were genotypically compared by pulsed-field gel electrophoresis of XbaI-macrorestricted chromosomal DNA. Altogether, these methods showed that 40 of the 41 PSE-1 producers were actually the progeny of a single epidemic S. typhimurium strain lysotype DT104. Isolates of that strain were found to harbor RepFIC virulence plasmids with somewhat different restriction profiles, but which all carried the bla(PSE-1) gene. Of these virulence/resistance plasmids, 15 were transmissible to Escherichia coli. TEM-1-producing S. typhimurium displayed much greater genotypic and plasmidic diversities, suggesting the acquisition of the bla(TEM-1) gene from multiple bacterial sources by individual strains. In agreement with this, 32 of the 35 S. typhimurium plasmids encoding TEM-1 were found to be conjugative. These data show that development of amoxicillin resistance among Salmonella, especially in serovar Typhimurium, results from both gene transfers and strain dissemination.

Amoxicillin↗
PubMed1999

In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM.

During a 6-month period, 21 pairs of Pseudomonas aeruginosa isolates susceptible (pretherapy) and resistant (posttherapy) to antipseudomonal beta-lactam antibiotics were isolated from hospitalized patients. In vivo emergence of beta-lactam resistance was associated with the overexpression of AmpC beta-lactamase in 10 patients. In the other 11 patients, the posttherapy isolates produced only low, basal levels of beta-lactamase and had increased levels of resistance to a variety of non-beta-lactam antibiotics (e.g., quinolones, tetracyclines, and trimethoprim) compared with the levels of beta-lactamase production and resistance of their pretherapy counterparts. These data suggested the involvement of the MexA-MexB-OprM active efflux system in the multidrug resistance phenotype of the posttherapy strains. Immunoblotting of the outer membrane proteins of these 11 bacterial pairs with a specific polyclonal antibody raised against OprM demonstrated the overexpression of OprM in all the posttherapy isolates. To determine whether mutations in mexR, the regulator gene of the mexA-mexB-oprM efflux operon, could account for the overproduction of the efflux system, sequencing experiments were carried out with the 11 bacterial pairs. Eight posttherapy isolates were found to contain insertions or deletions that led to frameshifts in the coding sequences of mexR. Two resistant strains had point mutations in mexR that yielded single amino acid changes in the protein MexR, while another strain did not show any mutation in mexR or in the promoter region upstream of mexR. Introduction of a plasmid-encoded wild-type mexR gene into five posttherapy isolates partially restored the susceptibility of the bacteria to selected antibiotics. These results indicate that in the course of antimicrobial therapy multidrug-resistant active efflux mutants overexpressing the MexA-MexB-OprM system may emerge as a result of mutations in the mexR gene.

Anti-Bacterial Agents↗
PubMed1999

Uptake of pyocin S3 occurs through the outer membrane ferripyoverdine type II receptor of Pseudomonas aeruginosa.

Pyocin S3 was found to kill exclusively Pseudomonas aeruginosa isolates producing type II pyoverdine (exemplified by strain ATCC 27853). Killing was specifically inhibited by addition of type II ferripyoverdine. All Tn5 mutants resistant to pyocin S3 were defective for pyoverdine-mediated iron uptake and failed to produce an 85-kDa iron-repressed outer membrane protein. We conclude that this protein is probably the type II ferripyoverdine receptor that is used by pyocin S3 to gain entry into the cell.

Bacterial Outer Membrane Proteins↗
PubMed1997

Differential selection of multidrug efflux systems by quinolones in Pseudomonas aeruginosa.

Resistance mechanisms selected after in vitro exposure to 12 quinolones were analyzed for Pseudomonas aeruginosa. Efflux-type mutants were predominant. Quinolones differed in their ability to select a particular efflux system. While the newer fluoroquinolones favored the MexCD-OprJ system, the older quinolones selected exclusively the MexEF-OprN or MexAB-OprM systems. A protonable C-7 substituent in combination with a C-6 fluorine atom is a structural determinant of quinolones involved in efflux pump substrate specificity.

Anti-Infective Agents↗
PubMed1997

Dechlorination of Atrazine by a Rhizobium sp. Isolate.

A Rhizobium sp. strain, named PATR, was isolated from an agricultural soil and found to actively degrade the herbicide atrazine. Incubation of PATR in a basal liquid medium containing 30 mg of atrazine liter(sup-1) resulted in the rapid consumption of the herbicide and the accumulation of hydroxyatrazine as the only metabolite detected after 8 days of culture. Experiments performed with ring-labeled [(sup14)C]atrazine indicated no mineralization. The enzyme responsible for the hydroxylation of atrazine was partially purified and found to consist of four 50-kDa subunits. Its synthesis in PATR was constitutive. This new atrazine hydrolase demonstrated 92% sequence identity through a 24-amino-acid fragment with atrazine chlorohydrolase AtzA produced by Pseudomonas sp. strain ADP.

Journal Article↗
PubMed1997

Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa.

Testosterone (a strongly hydrophobic steroid) and testosterone hemisuccinate (a negatively charged derivative) were used as probes to investigate alterations in the outer membrane of Pseudomonas aeruginosa. Diffusion rates of the steroids across the lipid bilayer were measured by coupling the influx of these compounds to their subsequent oxidation by an intracellular delta1-dehydrogenase enzyme. Wild-type cells of P. aeruginosa (strain PAO1) were found to be 25 times more permeable to testosterone than to testosterone hemisuccinate. The uptake of the latter compound appeared to be partially dependent on the external pH, thus suggesting a preferential diffusion of the uncharged protonated form across the cell envelope. Using various PAO mutants, we showed that the permeation of steroids was not affected by overexpression of active efflux systems but was increased up to 5.5-fold when the outer membrane contained defective lipopolysaccharides or lacked the major porin OprF. Such alterations in the hydrophobic uptake pathway were not, however, associated with an enhanced permeability of the mutants to the small hydrophilic molecule N,N,N',N'-tetramethyl-p-phenylene diamine. Thirty-six agents were also assayed for their ability to damage the cell surface of strain PAO1, using testosterone as a probe. Polymyxins, rBPI23, chlorhexidine, and dibromopropamidine demonstrated the strongest permeabilizing activities on a molar basis in the presence of 1 mM MgCl2. These amphiphilic polycations increased the transmembrane diffusion of testosterone up to 50-fold and sensitized the PAO1 cells to hydrophobic antibiotics. All together, these data indicated that the steroid uptake assay provides a direct and accurate measurement of the hydrophobic uptake pathway in P. aeruginosa.

Anti-Bacterial Agents↗
PubMed1997

Genotypic analysis of Burkholderia cepacia isolates from 13 French cystic fibrosis centers.

Burkholderia cepacia has been involved in outbreaks of pulmonary infection among patients with cystic fibrosis (CF), and the spread of a highly transmissible clone has been reported throughout the United Kingdom and Canada. These data prompted a DNA-based typing study of the strains recovered in French CF centers. Ninety-five isolates recovered from 71 patients attending 13 CF centers in 9 regions of France were characterized by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Twenty-one genotypes were identified among the 95 isolates, and the results of RAPD and PFGE were concordant for 89 isolates (94%). Cross-colonization was demonstrated in 7 of the 13 CF centers. The investigation of serial isolates showed that most chronically colonized patients harbored a single B. cepacia strain. A geographically clustered distribution of B. cepacia genotypes was observed, except for one genotype, which was detected in four regions but was proven to be different from the genotype of the British-Canadian highly transmissible strain. The present study confirms the ability of B. cepacia to spread among CF communities in France and the importance of epidemiological surveys in the institution of prevention policies.

Bacterial Typing Techniques↗
PubMed1996

Molecular analysis of the replication elements of the broad-host-range RepA/C replicon.

RepA/C is a replicon specific to the IncA/C incompatibility group of plasmids and was isolated recently from plasmid RA1. The sequence of this autoreplicative region was established; it contains 13 repeats, suggesting that the replicon uses iterons to control its copy number. The sequence contains two ORFs, one potentially coding for a 33-kDa protein (ORF1) and a second potentially coding for a 14-kDa protein (ORF2) (Llanes et al., 1994b). In this work, using an in vitro transcription/translation system, we detected a polypeptide whose size corresponded well to that of the deduced product of ORF1. Deletion and insertion mutation analysis showed that ORF1 is essential for replication; it encodes an initiator protein (called RepA). ORF2 was not essential for replication in Escherichia coli and its function remains to be determined. Using complementation experiments, the replication origin (ori) of RepA/C was defined. The ori was located in a 600-bp fragment downstream from repA, containing 10 direct repeats. To study the control of repA expression, a transcriptional fusion PrepA::lacZ was constructed. Its analysis showed that repA is transcriptionally autoregulated as are most repA genes of replicons controlled by iterons.

Cloning, Molecular↗
PubMed1996

Multidrug efflux in intrinsic resistance to trimethoprim and sulfamethoxazole in Pseudomonas aeruginosa.

Pseudomonas aeruginosa possesses at least two multiple drug efflux systems which are defined by the outer membrane proteins OprM and OprJ. We have found that mutants overexpressing OprM were two- and eightfold more resistant than their wild-type parent to sulfamethoxazole (SMX) and trimethoprim (TMP), respectively. For OprJ-overproducing strains, MICs of TMP increased fourfold but those of SMX were unchanged. Strains overexpressing OprM, but not those overexpressing OprJ, became hypersusceptible to TMP and SMX when oprM was inactivated. The wild-type antibiotic profile could be restored in an oprM mutant by transcomplementation with the cloned oprM gene. These results demonstrate that the mexABoprM multidrug efflux system is mainly responsible for the intrinsic resistance of P. aeruginosa to TMP and SMX.

Anti-Bacterial Agents↗
PubMed1996

Comamonas testosteroni 3-ketosteroid-delta 4(5 alpha)-dehydrogenase: gene and protein characterization.

Comamonas testosteroni delta 4(5 alpha)- and delta1-dehydrogenases [delta4(5alpha)- and delta1DH] are key enzymes in the degradation of steroids having an A:B ring fusion in a trans configuration. We previously reported the isolation of the delta1dh gene (P. Plesiat, M. Grandguillot, S. Harayama, S. Vragar, and Y. Michel Briand, J. Bacteriol. 173:7219-7227, 1991). In this study, the gene encoding delta 4(5 alpha)DH was cloned in Escherichia coli on a 16-kbp BamHI fragment by screening a genomic bank of C. testosteroni ATCC 17410 with a probe derived from delta1dh. Subcloning experiments in plasmid pUC19 mapped delta 4(5 alpha)dh immediately downstream of delta1dh. The enzyme was overexpressed 18-fold in cells of E. coli JM109 carrying a 2.5-kbp cloned fragment (plasmid pXE25). However, much higher levels of enzymatic activity (264-fold) were obtained in Pseudomonas putida KT2440, using pMMB208 as an expression vector. Studies with crude lysates of KT2440 showed that delta4(5alpha)DH exhibits higher specificity and higher activity toward delta l-androstene-3,17-dione than toward the saturated derivative 5 alpha-androstane-3,17-dione. The reaction was found to be irreversible and to use efficiently typical flavoprotein electron acceptors; optimal conditions for the enzyme activity were pH 8 and 40 degrees C. Analysis of the nucleotide sequence of the insert of pXE25 revealed an open reading frame of 1,593 bp preceded by a putative ribosome-binding site and followed by a potential transcription terminator. The amino acid sequence of the deduced peptide showed a typical flavin adenine dinucleotide-binding site in its N-terminal region, confirming the flavoproteinic structure of delta 4(5 alpha)DH. The predicted molecular mass was consistent with that of the enzyme expressed in a T7 polymerase system (60 kDa). Alignment between delta 4(5 alpha)dh and delta1dh indicated that both genes, though coding for functionally related enzymes, do not derive from a common ancestor.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗