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1.  Evaluation and Updating of the Osiris Expert System for Identification of Escherichia coli β-Lactam Resistance Phenotypes 
Journal of Clinical Microbiology  2005;43(4):1846-1850.
Osiris is a video zone size reader for disk diffusion tests that includes a built-in extended expert system (EES). We evaluated the efficacy of the Osiris EES for the identification of β-lactam susceptibility phenotypes of Escherichia coli isolates. Fifteen β-lactam agents and three β-lactam-β-lactamase inhibitor combinations were tested by the disk diffusion method against 50 E. coli strains with well-characterized resistance mechanisms. The strains were screened for the production of extended-spectrum β-lactamase (ESBL) by the double-disk synergy test using a disk of amoxicillin-clavulanic acid with disks of the extended-spectrum cephalosporins and aztreonam. Overall, the EES accurately identified the phenotype for 78% of the strains, indicated an inexact phenotype for 17% of the strains, and could not find a matching phenotype for the remaining 5% of the strains. The percentage of correct identification for each resistance mechanism was 100% for inhibitor-resistant TEM and for TEM plus cephalosporinase, 88.9% for TEM and for ESBL, 70.8% for cephalosporinase overproduction, and 25% for oxacillinase. The main cause of discrepancy was the misidentification of oxacillinase as inhibitor-resistant TEM. The conventional double-disk synergy test failed to detect ESBL production in two strains (one producing VEB-1 and one producing CTX-M-14), but synergy between cefepime and amoxicillin-clavulanic acid was visible after the distance between the disks was reduced to 20 mm. After the interpretative guidelines of the EES were updated according to our results, the percentage of correct phenotype identification increased from 78 to 96%.
PMCID: PMC1081361  PMID: 15815007
2.  Fluorescence Assay for Studying the Ability of Macrolides To Induce Production of Ribosomal Methylase 
A screening assay to test the inducing capacity of macrolides by fusing the attenuator of the inducible erm(B) gene from Streptococcus pneumoniae HM28 with the gfpmut1 gene has been designed. Fluorescence was detected under UV light around disks impregnated with inducer macrolides (erythromycin or azithromycin) but not with noninducer ketolides. Induction could be quantified by fluorometry.
PMCID: PMC127294  PMID: 12069987
3.  Macrolide Resistance Gene mreA of Streptococcus agalactiae Encodes a Flavokinase 
The mreA gene from Streptococcus agalactiae COH31 γ/δ, resistant to macrolides and clindamycin by active efflux, has recently been cloned in Escherichia coli, where it was reported to confer macrolide resistance (J. Clancy, F. Dib-Hajj, J. W. Petitpas, and W. Yuan, Antimicrob. Agents Chemother. 41:2719–2723, 1997). Cumulative data suggested that the mreA gene was located on the chromosome of S. agalactiae COH31 γ/δ. Analysis of the deduced amino acid sequence of mreA revealed significant homology with several bifunctional flavokinases/(flavin adenine dinucleotide (FAD) synthetases, which convert riboflavin to flavin mononucleotide (FMN) and FMN to FAD, respectively. High-performance liquid chromatography experiments showed that the mreA gene product had a monofunctional flavokinase activity, similar to that of RibR from Bacillus subtilis. Sequences identical to those of the mreA gene and of a 121-bp upstream region containing a putative promoter were detected in strains of S. agalactiae UCN4, UCN5, and UCN6 susceptible to macrolides. mreA and its allele from S. agalactiae UCN4 were cloned on the shuttle vector pAT28. Both constructs were introduced into E. coli, where they conferred a similar two- to fourfold increase in the MICs of erythromycin, spiramycin, and clindamycin. The MICs of a variety of other molecules, including crystal violet, acriflavin, sodium dodecyl sulfate, and antibiotics, such as certain cephalosporins, chloramphenicol, doxycycline, nalidixic acid, novobiocin, and rifampin, were also increased. In contrast, resistance to these compounds was not detected when the constructs were introduced into E. faecalis JH2–2. In conclusion, the mreA gene was probably resident in S. agalactiae and may encode a metabolic function. We could not provide any evidence that it was responsible for macrolide resistance in S. agalactiae COH31 γ/δ; broad-spectrum resistance conferred by the gene in E. coli could involve multidrug efflux pumps by a mechanism that remains to be elucidated.
PMCID: PMC90643  PMID: 11451686

Results 1-3 (3)