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J Bille

Publications and source records attributed to J Bille.

At least 55 records · Page 3Linked to original sources
PubMed1999

The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents.

The resistance mechanisms to azole antifungal agents were investigated in this study with two pairs of Candida glabrata clinical isolates recovered from two separate AIDS patients. The two pairs each contained a fluconazole-susceptible isolate and a fluconazole-resistant isolate, the latter with cross-resistance to itraconazole and ketoconazole. Since the accumulation of fluconazole and of another unrelated substance, rhodamine 6G, was reduced in the azole-resistant isolates, enhanced drug efflux was considered as a possible resistance mechanism. The expression of multidrug efflux transporter genes was therefore examined in the azole-susceptible and azole-resistant yeast isolates. For this purpose, C. glabrata genes conferring resistance to azole antifungals were cloned in a Saccharomyces cerevisiae strain in which the ATP binding cassette (ABC) transporter gene PDR5 was deleted. Three different genes were recovered, and among them, only C. glabrata CDR1 (CgCDR1), a gene similar to the Candida albicans ABC transporter CDR genes, was upregulated by a factor of 5 to 8 in the azole-resistant isolates. A correlation between upregulation of this gene and azole resistance was thus established. The deletion of CgCDR1 in an azole-resistant C. glabrata clinical isolate rendered the resulting mutant (DSY1041) susceptible to azole derivatives as the azole-susceptible clinical parent, thus providing genetic evidence that a specific mechanism was involved in the azole resistance of a clinical isolate. When CgCDR1 obtained from an azole-susceptible isolate was reintroduced with the help of a centromeric vector in DSY1041, azole resistance was restored and thus suggested that a trans-acting mutation(s) could be made responsible for the increased expression of this ABC transporter gene in the azole-resistant strain. This study demonstrates for the first time the determinant role of an ABC transporter gene in the acquisition of resistance to azole antifungals by C. glabrata clinical isolates.

ATP-Binding Cassette Transporters↗
PubMed1999

Division of Listeria monocytogenes serovar 1/2a strains into two groups by PCR and restriction enzyme analysis.

Altogether, 100 strains of Listeria monocytogenes serovar 1/2a isolated from humans, animals, food, and the environment were typed by a combination of PCR and restriction enzyme analysis (REA). A PCR product of 2,916 bp, containing the downstream end of the gene inlA (955 bp), the space between inlA and inlB (85 bp), and 1,876 bp of the gene inlB, was cleaved with the enzyme AluI, and the fragments generated were separated by gel electrophoresis. By this method two different cleavage patterns were obtained. Seventy of the 100 strains shared one restriction profile, and the remaining 30 strains shared the second one. No relation was found between the types differentiated by PCR-REA and the origins of the strains.

Animals↗
PubMed1999

Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus.

The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of the mecA gene for the detection of methicillin resistance in Staphylococcus aureus. A total of 120 methicillin-susceptible S. aureus (MSSA) and 80 methicillin-resistant S. aureus (MRSA) isolates, defined by the absence or presence of the mecA gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates.

Antibodies, Monoclonal↗
PubMed1999

[Hand infections].

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Anti-Bacterial Agents↗
PubMed1999

Listeria monocytogenes subtypes associated with mortality among fallow deer (Dama dama).

Different subtypes of Listeria monocytogenes were isolated from various animal and environmental samples during an episode of increased mortality on a fallow deer (Dama dama) farm. During a 4-wk period, six fallow deer died, including four does, one fawn, and one adult buck. Prior to death, one of the does had exhibited central nervous system signs characteristic of listeriosis. Postmortem examination of the six deer showed no histologic changes typical of listeriosis, although inflammatory changes were present in several organs. Different subtypes of L. monocytogenes were isolated from brain samples from six deer, from fodder and soil from the deer feeding area, and from faces of some healthy animals on the farm. Listeria monocytogenes, which was frequently isolated in the environment of the farm, was considered the probable major cause of mortality in these fallow deer.

Animal Feed↗
PubMed1998

Multiple resistance mechanisms to azole antifungals in yeast clinical isolates.

The use of antifungal agents, especially the azole class, has increased in parallel with a higher incidence of fungal infections, particularly in immunocompromised patients. This situation has favored the appearance of Candida species, prominent among them C. albicans and C. globrata, with acquired resistance to these agents. This review focuses on the latest developments in investigations of molecular mechanisms contributing to azole resistance. Multiple resistance mechanisms have been described that can coexist in resistant clinical isolates. Understanding resistance mechanisms is of value not only for the design of new antifungal agents but also the development of strategies of overcome or delay the emergence of resistance.

Journal Article↗
PubMed1998

Comparison of PCR-based DNA fingerprinting techniques for the identification of Listeria species and their use for atypical Listeria isolates.

Four PCR-based DNA fingerprinting techniques were compared for their ability to identify at the species level a heterogeneous collection of isolates belonging to the six valid Listeria species. 16S rDNA-RFLP analysis identified all species and 16S rDNA-SSCP analysis identified almost all species. Also, isolates with unusual biochemical characteristics and/or unusual antigenic composition could be identified correctly. rRNA-intracistronic length polymorphism analysis suffered from high intraspecific variability, a limited number of fragments per profile, and small length differences between the spacers of different species. tRNA-intergenic length polymorphism analysis resulted in identification of all isolates but one, when fluorescent DNA capillary electrophoresis was used such that fragment length differences of 1 bp could be resolved. The four techniques yielded comparable results relevant to the taxonomy of Listeria. They all indicate a high degree of genetic relatedness between L. innocua and L. welshimeri, homogeneity of L. grayi, distinct but clear relatedness of L. grayi to the other Listeria species, a clear distinction between the two subspecies of L. ivanovii, and a clear distinction between Listeria isolates and isolates from closely related taxa or from species which are phenotypically difficult to distinguish from Listeria. New sequence determination of the 16S rRNA gene was necessary to obtain sequences in accordance with the findings of 16S rDNA-RFLP analysis.

DNA Fingerprinting↗
PubMed1998

Typing methods to approach Pneumocystis carinii genetic heterogeneity.

The study of the genetic heterogeneity of P. carinii is complicated by the lack of an in vitro culture system, as well as by the likely occurrence of co-infections with several special forms or types in a single host. Karyotyping and multilocus enzyme electrophoresis are useful for studies at the evolutionary level. However, these methods require a large number of cells, which prevents their use for the special form infecting humans. DNA sequence analysis of genomic regions is useful to study P. carinii diversity, both at the evolutionary and epidemiological levels. To type the special form specific to humans, several methods are currently used to detect polymorphism in PCR products of polymorphic regions of the genome: DNA sequencing, type-specific hybridisations, and single-strand conformation polymorphism. All these methods still need evaluation. The frequency of potential co-infections in humans determined by these various methods is different. The differences could be due to methodological problems or to real variations between patient populations, geographical locations and/or prophylaxis regimens. In the future, elucidating the population structure of P. carinii and the frequency of potential co-infections is going to be crucial for a better understanding of its epidemiology, and thus for a better prevention of P. carinii pneumonia in humans.

DNA, Fungal↗
PubMed1998

Prospective randomized comparison of imipenem-cilastatin and piperacillin-tazobactam in nosocomial pneumonia or peritonitis.

Nosocomial pneumonia and acute peritonitis may be caused by a wide array of pathogens, and combination therapy is often recommended. We have previously shown that imipenem-cilastatin monotherapy was as efficacious as the combination of imipenem-cilastatin plus netilmicin in these two settings. The efficacy of imipenem-cilastatin is now compared to that of piperacillin-tazobactam as monotherapy in patients with nosocomial pneumonia or acute peritonitis. Three hundred seventy one patients with nosocomial pneumonia or peritonitis were randomly assigned to receive either imipenem-cilastatin (0.5 g four times a day) or piperacillin-tazobactam (4.5 g three times a day). Three hundred thirteen were assessable (154 with nosocomial pneumonia and 159 with peritonitis). For nosocomial pneumonia, clinical-failure rates in the piperacillin-tazobactam group (13 of 75 [17%]) and in the imipenem-cilastatin group (23 of 79 [29%]) were similar (P = 0.09), as were the numbers of deaths due to infection (6 in the imipenem-cilastatin group [8%], 7 in the piperacillin-tazobactam group [9%]) (P = 0.78). For acute peritonitis, clinical success rates were comparable (piperacillin-tazobactam, 72 of 76 [95%]; imipenem-cilastatin, 77 of 83 [93%]). For infections due to Pseudomonas aeruginosa, 45 patients had nosocomial pneumonia (21 in the piperacillin-tazobactam group and 24 in the imipenem-cilastatin group) and 10 had peritonitis (5 in each group). In the patients with nosocomial pneumonia, clinical failure was less frequent in the piperacillin-tazobactam group (2 of 21 [10%]) than in the imipenem-cilastatin [corrected] group (12 of 24 [50%]) (P = 0.004). Bacterial resistance to allocated regimen was the main cause of clinical failure (1 in the piperacillin-tazobactam group and 12 in the imipenem-cilastatin group). For the patients with peritonitis, no difference in clinical outcome was observed (five of five cured in each group). The overall frequencies of adverse events related to treatment in the two groups were similar (24 in the piperacillin-tazobactam group, 22 in the imipenem-cilastatin group). Diarrhea was significantly more frequent in the piperacillin-tazobactam group (10 of 24) than in the imipenem-cilastatin group (2 of 22). This study suggests that piperacillin-tazobactam monotherapy is at least as effective and safe as imipenem-cilastatin monotherapy in the treatment of nosocomial pneumonia or peritonitis. In P. aeruginosa pneumonia, piperacillin-tazobactam achieved a better clinical efficacy than imipenem-cilastatin, due to reduced development of microbiological resistance. Tolerance was comparable, with the exception of diarrhea, which was more frequent with piperacillin-tazobactam.

Acute Disease↗
PubMed1998

Amino acid substitutions in the cytochrome P-450 lanosterol 14alpha-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents.

The cytochrome P-450 lanosterol 14alpha-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance of C. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378-2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression in Saccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was designed. This selection, which was coupled with a test of susceptibility to the azole derivatives fluconazole, ketoconazole, and itraconazole, enabled the detection of mutations in different cloned CYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives. This selection enabled the detection of five different mutations in the cloned CYP51A1 genes which correlated with the occurrence of azole resistance in clinical C. albicans isolates. These mutations were as follows: replacement of the glycine at position 129 with alanine (G129A), Y132H, S405F, G464S, and R467K. While the S405F mutation was found as a single amino acid substitution in a CYP51A1 gene from an azole-resistant yeast, other mutations were found simultaneously in individual CYP51A1 genes, i.e., R467K with G464S, S405F with Y132H, G129A with G464S, and R467K with G464S and Y132H. Site-directed mutagenesis of a wild-type CYP51A1 gene was performed to estimate the effect of each of these mutations on resistance to azole derivatives. Each single mutation, with the exception of G129A, had a measurable effect on the affinity of the target enzyme for specific azole derivatives. We speculate that these specific mutations could combine with the effect of multidrug efflux transporters in the clinical isolates and contribute to different patterns and stepwise increases in resistance to azole derivatives.

Amino Acid Sequence↗
PubMed1998

Rapid detection of Candida albicans in clinical samples by DNA amplification of common regions from C. albicans-secreted aspartic proteinase genes.

Laboratory diagnosis based on genomic amplification methods such as PCR may provide an alternative and more sensitive method than conventional culture for the early detection of deep-seated candidiasis, an increasing cause of morbidity and mortality among immunocompromised patients. A novel method of DNA extraction from clinical samples based on treatment with proteinase K and isolation of DNA on a silica membrane was developed. The targets used for DNA amplification were the Candida albicans-secreted aspartic proteinase (SAP) genes, a multiple-gene family of at least seven members in C. albicans. A single pair of primers was designed in order to detect six of these SAP genes and, subsequently, to increase the sensitivity of the test. Detection of the PCR product by enzyme-linked immunosorbent assay was found to be as sensitive as Southern blotting with an SAP-labeled probe. The sensitivity of the assay was 1 cell/ml from serially diluted Candida cultures and 1 to 4 cells/ml from seeded blood specimens. The sensitivity and specificity of the present assay were tested in a retrospective study performed blindly with 156 clinical samples and were 100 and 98%, respectively, compared with the results of culture. For the subset of blood culture samples (n = 124), the sensitivity and the specificity were 100%. The two false-positive PCR samples came from patients treated with azole antifungal agents, indicating that PCR was probably able to detect damaged organisms that could not be recovered by culture.

Aspartic Acid Endopeptidases↗
PubMed1997

Evaluation of the susceptibility of pathogenic Candida species to fluconazole. Fluconazole Global Susceptibility Study Group.

A fluconazole 25 microg disk diffusion test was used to test 2230 consecutively isolated Candida strains from 42 different hospital laboratories in 23 countries. Ninety seven percent of 1634 Candida albicans isolates and 83.4% of 596 non-Candida albicans isolates were susceptible to fluconazole, applying the proposed breakpoints (> or = 26 mm for susceptible strains and 18-25 mm for dose-dependent susceptible strains). This is the first hospital laboratory study to evaluate a large number and wide range of sequential Candida isolates from patients with all types of hospital infections. The fluconazole disk diffusion test appears to be a low-cost, reproducible, and accurate means of assessing the in vitro susceptibility of Candida isolates.

Antifungal Agents↗
PubMed1997

Enterococcus cecorum septicemia in a malnourished adult patient.

Enterococcus cecorum, a species typically isolated from chicken, pigs, calves, horses, ducks, cats, dogs, and canaries, was isolated from the blood of a patient with severe septicemia. The isolate was identified by conventional biochemical tests. Identity as Enterococcus cecorum was confirmed by SDS-PAGE analysis of whole cell protein. This is the first report of the isolation of Enterococcus cecorum in a clinical setting.

Adult↗
PubMed1997

Typing of Austrian Listeria monocytogenes isolates by automated laser fluorescence analysis of randomly amplified polymorphic DNA.

We used automated laser fluorescence analysis of randomly amplified polymorphic DNA (RAPD-Alfa) to study the epidemiology of listeriosis in western Austria. There were no discrepancies between RAPD-Alfa patterns and serotypes found in 18 food isolates and 18 clinical isolates. The results of our study suggest that the food isolates typed were not at the origin of the human cases in western Austria. Using RAPD-Alfa, it was possible to link 9 out of 16 "sporadic" Listeria infections (mother-child cases counted as one) to the occurrence of other cases. Our results underline the necessity of epidemiological clarification of listeriosis cases as a prerequisite for specific preventive measures by public health services (e.g. confiscation of contaminated food products, issue of public warnings). To establish the chain of infection, more is needed than just speciation of bacteria in incriminated food products. Automated laser fluorescence analysis of randomly amplified polymorphic DNA seems a suitable, easy and rapid method for the typing of Listeria monocytogenes strains.

Adult↗
PubMed1997

Development of a molecular typing method for Pneumocystis carinii sp.f. hominis.

We are developing a typing method for Pneumocystis carinii sp.f. hominis consisting in the PCR amplification of four variable regions of the genome from bronchoalveolar lavages, followed by the detection of their variation by the single-strand conformation polymorphism technique (SSCP). Most samples of each region from eleven unrelated patients showed two SSCP bands. Six patients were probably infected by a single strain since they showed a single sequence morph of each region. The combinations of the four sequence morphs of these patients were all different so that six different strains were distinguished. Other samples generated three or four SSCP bands which were found to correspond to the presence of two sequence morphs, possibly due to co-infections. Five patients could have been co-infected since they showed two sequence morphs of at least one of the regions. The combinations of sequence morphs of the possible co-infecting strains were different from all other combinations, except that three possible strains in two patients might have been present in other patients. Two BALs from the same patient, which were collected at an interval of 2.5 weeks, gave the same pattern for each of the four regions suggesting that these regions of the genome are stable. These results suggest a great diversity of P. carinii sp.f. hominis strains. Thus, the method should be suitable for epidemiological studies.

Mycological Typing Techniques↗