Linezolid, the first oxazolidinone to be introduced in clinical practice, has broad activity against important multi-drug-resistant Gram-positive pathogens, including meticillin-resistant staphylococci and vancomycin-resistant enterococci. Linezolid inhibits protein synthesis by binding to the peptidyl transferase centre of the bacterial ribosome. Although resistance may be mediated by transferable genes such as cfr (and, crucially for enterococci, optrA), mutations in ribosomal proteins L3 and L4 and in the central loop of domain V of 23S rDNA1 constitute the main mechanism associated with linezolid resistance among clinically isolated staphylococci.

The activity of levofloxacin and ulifloxacin on biofilm formation and persistence was evaluated on microorganisms isolated from urinary double-J-stents. We analyzed 51 bacterial strains and their susceptibility to different antimicrobial classes was determined. We evaluated the bacterial ability to form biofilm and the effects of different concentrations of levofloxacin and ulifloxacin on bacterial adhesion and biofilm persistence. Most of the strains were biofilm producers with no relevant difference in biofilm production at 24 or 48 hours. The fluoroquinolones were able to prevent biofilm formation, but not to eradicate the preformed biofilm. On the basis of our data we advise that antibiotic prophylaxis with fluoroquinolones may be most helpful if given at the time of stent insertion and at high dosage.

Prulifloxacin, a new fluoroquinolone, is a prodrug whose active compound, ulifloxacin, is derived from its transformation after oral administration and intestinal absorption. Based on early pharmacokinetic and pharmacodynamic data, the following MIC breakpoints have tentatively been proposed: < or = 1 microg/ml, susceptible; 2 microg/ml, intermediate; and > or = 4 microg/ml, resistant. In this report, ulifloxacin MIC vs. zone diameter scattergrams and discrepancy rates were analyzed in 461 freshly isolated clinical strains (237 Enterobacteriaceae, 101 nonfermenters, and 123 Gram-positive bacteria). In agreement with the guidelines of the National Committee for Clinical Laboratory Standards, a modification of the error rate-bounded method was used to select disk diffusion test breakpoints. The following zone diameter breakpoints were chosen and are proposed herein for the interpretation of ulifloxacin disk (5 microg) test results: < or = 15 and > or = 19 mm for Enterobacteriaceae, < or = 16 and > or = 20 mm for nonfermenters, and < or = 14 and > or = 18 mm for Gram-positive bacteria. By applying these breakpoint values, no very major errors were detected, while major and minor errors were largely below the accepted discrepancy rates.

ABSTRACT The molecular genetics of macrolide resistance were analyzed in 49 clinical pneumococci (including an “atypical” bile-insoluble strain currently assigned to the new species Streptococcus pseudopneumoniae ) with efflux-mediated erythromycin resistance (M phenotype). All test strains had the mef gene, identified as mef (A) in 30 isolates and mef (E) in 19 isolates (including the S. pseudopneumoniae strain) on the basis of PCR-restriction fragment length polymorphism analysis. Twenty-eight of the 30 mef (A) isolates shared a pulsed-field gel electrophoresis (PFGE) type corresponding to the England 14 -9 clone. Of those isolates, 27 (20 belonging to serotype 14) yielded multilocus sequence type ST9, and one isolate yielded a new sequence type. The remaining two mef (A) isolates had different PFGE types and yielded an ST9 type and a new sequence type. Far greater heterogeneity was displayed by the 19 mef (E) isolates, which fell into 11 PFGE types, 12 serotypes (though not serotype 14), and 12 sequence types (including two new ones and an undetermined type for the S. pseudopneumoniae strain). In all mef (A) pneumococci, the mef element was a regular Tn 1207.1 transposon, whereas of the mef (E) isolates, 17 carried the mega element and 2 exhibited a previously unreported organization, with no PCR evidence of the other open reading frames of mega. The mef gene of these two isolates, which did not match with the mef (E) gene of the mega element (93.6% homology) and which exhibited comparable homology (91.4%) to the mef (A) gene of the Tn 1207.1 transposon, was identified as a novel mef gene variant and was designated mef (I). While penicillin-nonsusceptible isolates (three resistant isolates and one intermediate isolate) were all mef (E) strains, tetracycline resistance was also detected in three mef (A) isolates, due to the tet (M) gene carried by a Tn 916 -like transposon. A similar mechanism accounted for resistance in four of the five tetracycline-resistant isolates carrying mef (E), in three of which mega was inserted in the Tn 916 -like transposon, giving rise to the composite element Tn 2009 . In the fifth mef (E)-positive tetracycline-resistant isolate (the S. pseudopneumoniae strain), tetracycline resistance was due to the presence of the tet (O) gene, apparently unlinked to mef (E).

Fifteen Streptococcus pneumoniae clinical isolates with reduced fluoroquinolone susceptibility (defined as a ciprofloxacin MIC of > or = 4 microg/ml), all collected in Italy in 2000-2003, were typed and subjected to extensive molecular characterization to define the contribution of drug target alterations and efflux mechanisms to their resistance. Serotyping and pulsed-field gel electrophoresis analysis indicated substantial genetic unrelatedness among the 15 isolates, suggesting that the new resistance traits arise in multiple indigenous strains rather than through clonal dissemination. Sequencing of the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE demonstrated that point mutations producing single amino acid changes were more frequent in topoisomerase IV (parC mutations in 14 isolates and parE mutations in 13) than in DNA gyrase subunits (gyrA mutations in 7 isolates and no gyrB mutations observed). No isolate displayed a quinolone efflux system susceptible to carbonyl cyanide m-chlorophenylhydrazone; conversely, four-fold or greater MIC reductions in the presence of reserpine were observed in all 15 isolates with ethidium bromide, in 13 with ulifloxacin, in 9 with ciprofloxacin, in 5 with norfloxacin, and in none with five other fluoroquinolones. The effect of efflux pump activity on the level and profile of fluoroquinolone resistance in our strains was minor compared with that of target site modifications. DNA mutations and/or efflux systems other than those established so far might contribute to the fluoroquinolone resistance expressed by our strains. Susceptibility profiles to nonquinolone class antibiotics and resistance-associated phenotypic and genotypic characteristics were also determined and correlated with fluoroquinolone resistance. A unique penicillin-binding protein profile was observed in all five penicillin-resistant isolates, whereas the same PBP profile as S. pneumoniae R6 was exhibited by all six penicillin-susceptible isolates. This is the first attempt to molecularly characterize clinical isolates of S. pneumoniae with reduced susceptibility to fluoroquinolones emerging in Italy.