Manuela Prenna

Pharmacokinetics of Fluconazole in Normal Volunteers

The pharmacokinetic profile of fluconazole, after 100 mg i.v. infusion or oral administration of a single 50 mg or 150 mg dose, was investigated in 18 healthy volunteers. At a dose of 100 mg i.v., the half-life (t1/2 beta) was 29.73 +/- 8.05h. The mean residence time in the plasma was 27.56 +/- 5.98 h. The post-distributive volume V beta = 52.16 +/- 9.83 l, approximating that of total body water. Renal excretion accounted for 61.64 +/- 8.80% of the drug elimination after 48 h, with renal clearance Clr = 12.91 +/- 2.83 ml/min. Plasma clearance (Clp) was 21.03 +/- 5.07 ml/min. At oral doses of 50 and 150 mg the distribution and elimination of fluconazole resembled that following i.v. infusion. The peak levels in plasma at 2.5 h were 0.93 +/- 0.13 and 2.69 +/- 0.43 micrograms/ml, respectively. The large distribution volume, the long half-life and mean residence times, combined with a rapid absorption after oral administration, suggest that fluconazole will be effective at a wide range of body sites.

Analysis of different genetic traits and their association with biofilm formation in Staphylococcus epidermidis isolates from central venous catheter infections

The aim of the present study was to characterize clinical isolates of Staphylococcus epidermidis, one of the bacterial species most often implicated in foreign-body-associated infections, for their ability to form biofilms and for the presence of mecA and IS256 element. Sixty-seven Staphylococcus epidermidis clinical isolates, obtained from implantable medical devices, were investigated. Overall, 70% of the strains were positive for ica operon genes, 85% possessed atlE, and 46% contained aap. In 89% of the population, the Congo red agar test confirmed the correlation between the presence of ica genes and slime expression. Almost all of the strains could be classified as biofilm producers by both the crystal violet assay and microscopy. The bacterial population studied showed a very high frequency of strains positive for mecA as well as for the IS256 element. Although well-structured biofilms have been previously observed only in those strains possessing genes belonging to the ica operon, this study demonstrates that strains lacking specific biofilm-formation determinants can be isolated from catheters and can form a biofilm in vitro. Hence, different and yet-to-be identified factors may work together in the formation and organization of complex staphylococcal microbial communities and sustain infections associated with implanted medical devices.

SmaI macrorestriction analysis of Italian isolates of erythromycin-resistant Streptococcus pyogenes and correlations with macrolide resistance phenotypes.

High rates of erythromycin resistance among Streptococcus pyogenes strains have been reported in Italy in the last few years. In this study, 370 erythromycin-resistant (MIC, > or = 1 microg/mL) Italian isolates of this species obtained in 1997-1998 from throat swabs from symptomatic patients were typed by analyzing SmaI macrorestriction fragment patterns by pulsed-field gel electrophoresis (PFGE). Among the typable isolates (n = 341; the genomic DNA of the remaining 29 isolates was not restricted by SmaI), 48 distinct PFGE types were recognized, of which 31 were recorded in only one isolate (one-strain types). Fifty-two percent of typable isolates fell into three type clusters and 75% into six, suggesting that erythromycin-resistant group A streptococci circulating in Italy are polyclonal, but the majority of them probably derives from the spread of a limited number of clones. In parallel experiments, the 370 test strains were characterized for the macrolide resistance phenotype: 80 were assigned to phenotype cMLS, 89 to phenotype iMLS-A, 33 to phenotype iMLS-B, 11 to phenotype iMLS-C, and 157 to phenotype M. There was a close correlation between these phenotypic data and the genotypic results of PFGE analysis, the vast majority of the isolates assigned to individual PFGE classes belonging usually to a single phenotype of macrolide resistance. All of the 29 untypable isolates belonged to the M phenotype. Further correlations were observed with tetracycline resistance.

Analysis of meticillin-susceptible and meticillin- resistant biofilm-forming Staphylococcus aureus from catheter infections isolated in a large Italian hospital

Several characteristics were analysed in 37 Staphylococcus aureus isolates from nosocomial catheter infections: the PFGE profile after SmaI digestion of chromosomal DNA, the ability to form a biofilm on a polystyrene surface, antibiotic susceptibility patterns (penicillin, oxacillin, erythromycin, tetracycline, clindamycin, telithromycin, gentamicin, ciprofloxacin, quinupristin/dalfopristin, rifampicin, vancomycin and linezolid), and the presence of genetic determinants of antibiotic resistance and biofilm formation. All strains but three (92 %) were able to grow on a plastic surface as a biofilm. An almost complete association was found between phenotypes and genotypic traits of antibiotic resistance, whilst PFGE profiling showed the highly polyclonal composition of the set of strains under study. Sixteen isolates (43 %) were meticillin-resistant and were subjected to staphylococcal cassette chromosome mec (SCCmec) and cassette chromosome recombinase (ccr) complex type determination by multiplex PCR. Only a subgroup of six strains belonged to the archaic clone PFGE type and bore the SCCmec/ccrAB type I structure. Among the remaining strains some presented small rearrangements of the SCCmec/ccrAB genetic locus, whilst others could barely be traced back to a known structural type. These observations suggest that, at the local level and at a particular site of infection, S. aureus may show great genetic variability and escape the general rule of expansion of the S. aureus pandemic clones.

emm Gene distribution among erythromycin-resistant and -susceptible Italian isolates of Streptococcus pyogenes.

ABSTRACT The phenotypes and genetic determinants for macrolide resistance were determined for 167 erythromycin-resistant Streptococcus pyogenes strains. A cMLS phenotype was shown in 18% of the erythromycin-resistant strains, while inducible resistance was apparent in 31% and the M phenotype was apparent in 50%. The emm gene type of this set of resistant isolates and that of 48 erythromycin-sensitive isolates were determined. emm2 and emm48 were recorded only in the resistant strains of the M phenotype, while approximately all of the strains harboring the emm22 gene had the cMLS phenotype. More than 80% of the emm89-positive strains had the iMLS phenotype, and the same portion of emm4 strains presented the M phenotype. emm3 is recorded only among sensitive strains. The distribution of frequencies of the genetic determinant for the virulence factor M protein was significantly different both among organisms of different types of resistance and between resistant and sensitive populations of S. pyogenes under study.

Genetic Diversity of Cell-Invasive Erythromycin-Resistant and -Susceptible Group A Streptococci Determined by Analysis of the RD2 Region of the prtF1 Gene

ABSTRACT The RD2 region of the internalization-associated gene prtF1, which encodes the fibronectin-binding repeat domain type 2 of protein F1, plays a crucial role in the entry of group A streptococci (GAS) into epithelial cells. A molecular study of the variability of the RD2 region was carried out with 77 independent Italian GAS, 66 erythromycin resistant (ER) and 11 erythromycin susceptible (ES), which had previously been investigated for the association between erythromycin resistance and ability to enter human respiratory cells. The amplicons obtained from PCR analysis of the RD2 region were consistent with a number of RD2 repeats ranging from one to five, more frequently four (n = 30), three (n = 27), and one (n = 18). A new method to type cell-invasive GAS (RD2 typing) was developed by combining PCR analysis of the RD2 region and restriction analysis of PCR products with endonucleases HaeIII, DdeI, and HinfI. Overall, 10 RD2 types (a to j) were distinguished (all detected among the 66 ER isolates, four detected among the 11 ES isolates). Comparison and correlation of RD2 typing data with the genotype and phenotype of macrolide resistance and with data from PCR M typing and SmaI macrorestriction analysis allowed us to identify 41 different clones (31 among the 66 ER isolates and 10 among the 11 ES isolates). Three major clones accounted for 40% of the isolates (47% of ER strains). Some ES isolates appeared to be related to ER isolates with identical combinations of RD2 type and emm type. While simultaneous use of different typing methods is essential for a thorough investigation of GAS epidemiology, RD2 typing may be especially helpful in typing cell-invasive GAS.

Antibacterial Activity and Anti-Biofilm Effect of Chitosan against Strains of Streptococcus Mutans Isolated in Dental Plaque

Streptococcus mutans is the major cause of dental plaque and is often associated with biofilm formation. The aim of this study is to evaluate the activity of a hydrosoluble derivative of chitosan against S. mutans biofilms in vitro and in vivo. Strains of S. mutans were isolated from the dental plaque of 84 patients enrolled in the study. The antibacterial activity of chitosan was determined by broth microdilutions. The effect of chitosan at different concentrations and exposure times on S. mutans biofilms at different phases of development was assessed by a clinical study using the classical “4-day plaque regrowth” experiment in adult volunteers. The MIC values of chitosan were between 0.5 and 2 g/L. Compared to distilled water, the chitosan solution significantly decreased the vitality of plaque microflora (p≤0.05). Chlorhexidine, used as a positive control, reduced vitality even further. The results showed that S. mutans in the adhesion phase (4 h) was completely inhibited by chitosan at any concentration (0.1, 0.2, 0.5XMIC) or exposure time investigated (1, 15, 30, 60 min), while S. mutans at successive stages of accumulation (12–24 h) was inhibited only by higher concentrations and longer exposure times. These data confirm the effective action of chitosan against S. mutans biofilms.

A Linear Model for the Pharmacokinetics of Azithromycin in Healthy Volunteers

The pharmacokinetic profile of azithromycin, after oral ingestion of 500 mg, was determined in 10 healthy volunteers. Statistical and biochemical reason seemed to indicate a zero-order absorption of the drug. The disposition of azithromycin was described by a two-compartment model (plasma compartment and extravascular compartment) with elimination from the plasma compartment. The absorption process ends abruptly after a time T = 2.3 +/- 0.49 h, from the administration. The transfer rate constant from the plasma compartment to the extravascular compartment (k12 = 0.12 +/- 0.04 h-1) and the mean residence time of the drug in the extravascular compartment (MRT2 = 43.53 +/- 13.80 h) indicate a rapid and extensive distribution of azithromycin from the serum into the extravascular fluids. The results confirmed the efficacy of a single daily dose of 500 mg per os for clinical use.

Pharmacokinetics of cefadroxil after oral administration in humans.

The human oral pharmacokinetics of cefadroxil were studied in parallel at doses of 250, 500, and 1,000 mg in three groups of 10 healthy young male volunteers. Renal excretion of intact cefadroxil, accounted for 82, 79, and 77% of the above doses. Mean peak serum levels were dose linear: 9, 18, and 35 microgram/ml at 250, 500, and 1,000 mg, respectively. However, overall pharmacokinetics were linear only in the 250- to 500-mg dose range; apparent serum clearances were 10 liters/h, and true renal clearances were 9 and 8 liters/h at 250 and 500 mg. At 1,000 mg, apparent serum clearance dropped to about 7 liters/h, true renal clearance, dropped to 6 liters/h, and the area under the curve increased disproportionately. At 250 and 500 mg, mean half-life was about 1.2 h; at 1,000 mg, however, it was 1.6h. The nonlinear decrease in clearance could be related to saturation of active renal tubular secretion of cefadroxil between the 500- and 1,000-mg doses. Previous results indicating that cefadroxil has greater persistence than other oral cephalosporins such as cephalexin, cephradine, cefaclor were confirmed.

Biochemical Characterization of the THIN-B Metallo-β-Lactamase of Janthinobacterium lividum

ABSTRACT The THIN-B metallo-β-lactamase, a subclass B3 enzyme produced by the environmental species Janthinobacterium lividum, was overproduced in Escherichia coli by means of a T7-based expression system. The enzyme was purified (>95%) by two ion-exchange chromatography steps and subjected to biochemical analysis. The native THIN-B enzyme is a monomeric protein of 31 kDa. It exhibits the highest catalytic efficiencies with carbapenem substrates and cephalosporins, except for cephaloridine, which acts as a poor inactivator. Individual rate constants for inactivation by chelators were measured, suggesting that inactivation occurred by a mechanism involving formation of a ternary complex.