Effects of fluoroquinolones on bacterial adhesion and on preformed biofilm of strains isolated from urinary double J stents.

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.

A stochastic formulation of the gompertzian growth model for in vitro bactericidal kinetics: parameter estimation and extinction probability.

Time-kill curves have frequently been employed to study the antimicrobial effects of antibiotics. The relevance of pharmacodynamic modeling to these investigations has been emphasized in many studies of bactericidal kinetics. Stochastic models are needed that take into account the randomness of the mechanisms of both bacterial growth and bacteria-drug interactions. However, most of the models currently used to describe antibiotic activity against microorganisms are deterministic. In this paper we examine a stochastic differential equation representing a stochastic version of a pharmacodynamic model of bacterial growth undergoing random fluctuations, and derive its solution, mean value and covariance structure. An explicit likelihood function is obtained both when the process is observed continuously over a period of time and when data is sampled at time points, as is the custom in these experimental conditions. Some asymptotic properties of the maximum likelihood estimators for the model parameters are discussed. The model is applied to analyze in vitro time-kill data and to estimate model parameters; the probability of the bacterial population size dropping below some critical threshold is also evaluated. Finally, the relationship between bacterial extinction probability and the pharmacodynamic parameters estimated is discussed.

Antibiotic susceptibility and serotype distribution in Streptococcus pneumoniae circulating in Italy: results of the SEMPRE surveillance study (2000-2002).

During 2000-2002, 20 clinical microbiology centres collected 1623 Streptococcus pneumoniae isolates. Susceptibility to penicillin, amoxicillin, amoxicillin/clavulanic acid, cefaclor, cefuroxime, cefotaxime, clarithromycin, ciprofloxacin, levofloxacin, rifampicin and teicoplanin was determined locally by the Etest and/or by the microdilution method by three co-ordinating centres. Total resistance to penicillin increased from 15.2% to 16.1% and macrolide resistance increased from 37.9% to 43.7%. Overall, the most effective drugs (>99% susceptible strains) were amoxicillin, amoxicillin/clavulanic acid, levofloxacin and rifampicin. The most frequent serotypes were: 23F (15.8%), 3 (10.8%) 14 (9.1%), 19F (9.1%), 6B (7.2%), 19A (6.9%) and 6A (4.8%). In conclusion, penicillin and macrolide resistance is increasing in Italy, whilst fluoroquinolone currently remains active. The most common serotypes circulating are included in the heptavalent conjugate vaccine, with the exception of type 3.

Interpretive criteria for disk diffusion susceptibility testing of ulifloxacin, the active metabolite of prulifloxacin.

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.

In vitro antibacterial activities of AF 3013, the active metabolite of prulifloxacin, against nosocomial and community Italian isolates.

AF 3013, the active metabolite of prulifloxacin, was tested to determine its inhibitory and bactericidal activities against 396 nosocomial and 258 community Italian isolates. Compared with that of ciprofloxacin, its activity (assessed in MIC and minimal bactericidal concentration tests) was generally similar or greater against gram-positive bacteria and greater against gram-negative bacteria. In time-kill assays using selected isolates, its bactericidal activity was comparable to that of ciprofloxacin.

Differentiation of resistance phenotypes among erythromycin-resistant Pneumococci.

Laboratory differentiation of erythromycin resistance phenotypes is poorly standardized for pneumococci. In this study, 85 clinical isolates of erythromycin-resistant (MIC > or = 1 microg/ml) Streptococcus pneumoniae were tested for the resistance phenotype by the erythromycin-clindamycin double-disk test (previously used to determine the macrolide resistance phenotype in Streptococcus pyogenes strains) and by MIC induction tests, i.e., by determining the MICs of macrolide antibiotics without and with pre-exposure to 0.05 microg of erythromycin per ml. By the double-disk test, 65 strains, all carrying the erm(AM) determinant, were assigned to the constitutive macrolide, lincosamide, and streptogramin B resistance (cMLS) phenotype, and the remaining 20, all carrying the mef(E) gene, were assigned to the recently described M phenotype; an inducible MLS resistance (iMLS) phenotype was not found. The lack of inducible resistance to clindamycin was confirmed by determining clindamycin MICs without and with pre-exposure to subinhibitory concentrations of erythromycin. In macrolide MIC and MIC-induction tests, whereas homogeneous susceptibility patterns were observed among the 20 strains assigned to the M phenotype by the double-disk test, two distinct patterns were recognized among the 65 strains assigned to the cMLS phenotype by the same test; one pattern (n = 10; probably that of the true cMLS isolates) was characterized by resistance to rokitamycin also without induction, and the other pattern (n = 55; designated the iMcLS phenotype) was characterized by full or intermediate susceptibility to rokitamycin without induction turning to resistance after induction, with an MIC increase by more than three dilutions. A triple-disk test, set up by adding a rokitamycin disk to the erythromycin and clindamycin disks of the double-disk test, allowed the easy differentiation not only of pneumococci with the M phenotype from those with MLS resistance but also, among the latter, of those of the true cMLS phenotype from those of the iMcLS phenotype. While distinguishing MLS from M resistance in pneumococci is easily and reliably achieved, the differentiation of constitutive from inducible MLS resistance is far more uncertain and is strongly affected by the antibiotic used to test inducibility.

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.

In vitro activity of ketolides telithromycin and HMR 3004 against italian isolates of Streptococcus pyogenes and Streptococcus pneumoniae with different erythromycin susceptibility.

Two ketolides, telithromycin and HMR 3004, were evaluated for their in vitro activity against erythromycin-susceptible and -resistant strains of Streptococcus pyogenes and Streptococcus pneumoniae. On the basis of their resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics, erythromycin-resistant test strains were assigned to the constitutive resistance (cMLS) phenotype, the inducible resistance (iMLS) phenotype or the M phenotype. iMLS S. pyogenes strains were further subdivided into the three recently described subtypes iMLS-A, -B and -C. Telithromycin and HMR 3004 were uniformly and highly active against pneumococci (regardless of their susceptibility or resistance to erythromycin and/or penicillin), erythromycin-susceptible S. pyogenes and erythromycin-resistant S. pyogenes strains of the M phenotype (in which resistance is mediated by an efflux system) or iMLS-B or -C phenotype (in which resistance is mediated by a methylase encoded by the ermTR gene). Both ketolides were less active against erythromycin-resistant S. pyogenes strains with the cMLS phenotype or the iMLS-A subtype (where resistance is mediated by a methylase encoded by the ermAM gene), these strains ranging in phenotype from the upper limits of susceptibility to low-level resistant.

In vitro activity of levofloxacin against gram-positive bacteria.

The in vitro activity of levofloxacin was investigated against 256 clinical strains of four gram-positive genera (Staphylococcus, Streptococcus, Enterococcus, and Listeria). Ofloxacin and ciprofloxacin were used as comparators. Uniform susceptibility to levofloxacin was recorded among methicillin-susceptible staphylococci, streptococci other than Streptococcus agalactiae, regardless of their being susceptible, intermediate, or resistant to penicillin (S. pneumoniae) or erythromycin (S. pyogenes and S. pneumoniae), in enterococci other than Enterococcus faecalis and E. faecium, and in Listeria monocytogenes isolates. Moreover, 1 of 22 S. agalactiae isolates and 1 of 19 E. faecium isolates was resistant, and 2 of 19 were intermediate. Resistances to levofloxacin with MIC90s in the resistance range were only observed in methicillin-resistant staphylococci and E. faecalis isolates. In any case, the MICs of ofloxacin and ciprofloxacin were usually 2-4 times higher than those of levofloxacin. In time-kill assays using three test strains (a methicillin-susceptible Staphylococcus aureus isolate, a penicillin-susceptible Streptococcus pneumoniae isolate, and an E. faecalis isolate), the bactericidal activity of levofloxacin was greater than that of ciprofloxacin.

Phenotypes and genotypes of erythromycin-resistant Streptococcus pyogenes strains in Italy and heterogeneity of inducibly resistant strains.

A total of 387 clinical strains of erythromycin-resistant (MIC, >/=1 microg/ml) Streptococcus pyogenes, all isolated in Italian laboratories from 1995 to 1998, were examined. By the erythromycin-clindamycin double-disk test, 203 (52.5%) strains were assigned to the recently described M phenotype, 120 (31.0%) were assigned to the inducible macrolide, lincosamide, and streptogramin B resistance (iMLS) phenotype, and 64 (16.5%) were assigned to the constitutive MLS resistance (cMLS) phenotype. The inducible character of the resistance of the iMLS strains was confirmed by comparing the clindamycin MICs determined under normal testing conditions and those determined after induction by pregrowth in 0.05 microg of erythromycin per ml. The MICs of erythromycin, clarithromycin, azithromycin, josamycin, spiramycin, and the ketolide HMR3004 were then determined and compared. Homogeneous susceptibility patterns were observed for the isolates of the cMLS phenotype (for all but one of the strains, HMR3004 MICs were 0.5 to 8 microg/ml and the MICs of the other drugs were >128 microg/ml) and those of the M phenotype (resistance only to the 14- and 15-membered macrolides was recorded, with MICs of 2 to 32 microg/ml). Conversely, heterogeneous susceptibility patterns were observed in the isolates of the iMLS phenotype, which were subdivided into three distinct subtypes designated iMLS-A, iMLS-B, and iMLS-C. The iMLS-A strains (n = 84) were highly resistant to the 14-, 15-, and 16-membered macrolides and demonstrated reduced susceptibility to low-level resistance to HMR3004. The iMLS-B strains (n = 12) were highly resistant to the 14- and 15-membered macrolides, susceptible to the 16-membered macrolides (but highly resistant to josamycin after induction), and susceptible to HMR3004 (but intermediate or resistant after induction). The iMLS-C strains (n = 24) had lower levels of resistance to the 14- and 15-membered macrolides (with erythromycin MICs increasing two to four times after induction), were susceptible to the 16-membered macrolides (but resistant to josamycin after induction), and remained susceptible to HMR3004, also after induction. The erythromycin resistance genes in 100 isolates of the different groups were investigated by PCR. All cMLS and iMLS-A isolates tested had the ermAM (ermB) gene, whereas all iMLS-B and iMLS-C isolates had the ermTR gene (neither methylase gene was found in isolates of other groups). The M isolates had only the macrolide efflux (mefA) gene, which was also found in variable proportions of cMLS, iMLS-A, iMLS-B, and iMLS-C isolates. The three iMLS subtypes were easily differentiated by a triple-disk test set up by adding a josamycin disk to the erythromycin and clindamycin disks of the conventional double-disk test. Tetracycline resistance was not detected in any isolate of the iMLS-A subtype, whereas it was observed in over 90% of both iMLS-B and iMLS-C isolates.

In vitro antibacterial activity of trovafloxacin and five other fluoroquinolones.

The in vitro inhibitory and bactericidal activities of the investigational fluoroquinolone trovafloxacin were studied and compared with those of five other fluoroquinolones (ciprofloxacin, ofloxacin, pefloxacin, rufloxacin and sparfloxacin) against a wide range of clinical isolates from Italian hospitals. Against gram-positive bacteria, trovafloxacin was overall more active than the other antibiotics tested, including sparfloxacin, another gram-positive-oriented fluoroquinolone, and was active against all ciprofloxacin-resistant streptococci, enterococci, and listeriae, all ciprofloxacin-resistant Staphylococcus aureus isolates and most ciprofloxacin-resistant coagulase-negative staphylococci. Its antistaphylococcal activity was not affected by oxacillin resistance or susceptibility of the isolates, nor was its antipneumococcal activity affected by whether isolates were susceptible or resistant to penicillin. Against gram-negative bacteria, trovafloxacin retained a high potency mostly comparable with that of ciprofloxacin. Rufloxacin and pefloxacin were less active than the other fluoroquinolones against most test strains of both gram-positive and gram-negative organisms. Trovafloxacin minimal bactericidal concentrations usually equalled or exceeded by 2-4 times the minimal inhibitory concentration values, indicating that the compound is overall highly bactericidal.

Borderline methicillin-susceptible Staphylococcus aureus strains have more in common than reduced susceptibility to penicillinase-resistant penicillins.

Ten epidemiologically unrelated Staphylococcus aureus isolates with borderline levels of susceptibility to antistaphylococcal penicillinase-resistant penicillins (PRPs) were investigated together with appropriate S. aureus control strains. By a nitrocefin microplate assay, all borderline PRP-susceptible test strains were found to produce comparable amounts of beta-lactamase. Hydrolytic activity against another chromogenic substrate (PADAC) and against the PRPs was also demonstrated in membrane preparations from induced cells of 9 of the 10 borderline test strains. When bacterial membranes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two methicillin-inducible bands of about 32 and 31 kDa were detected, after Coomassie blue staining, in the membrane protein patterns of the same nine borderline test strains. By gel renaturation and zymographic detection of beta-lactamase activity, both methicillin-inducible membrane proteins were detected with nitrocefin as a substrate, whereas only one band (presumably the smaller protein) was detected with PADAC. With the remaining borderline test strain (a40), only the larger band was detected in the renatured gels with nitrocefin as a substrate. Plasmid DNA analysis revealed that the borderline susceptible test strains, again with the exception of a40, shared a 17.2-kb plasmid yielding four HindIII fragments of 7.0, 5.3, 3.5, and 1.4 kb. In Western blot (immunoblot) experiments using rabbit antiserum to penicillin-binding protein (PBP) 2a, test strain a40, which did not share a number of features characteristically associated with the other borderline test strains, was eventually shown to produce PBP 2a. Five other S. aureus strains, belonging to phage group 94/96, were found to display the borderline phenotype, including such distinguishing features as the membrane-associated PRP- and PADAC-hydrolyzing activity, the two methicillin-inducible membrane proteins, and the 17.2-kb plasmid. These results suggest that borderline susceptible S. aureus strains share more common features than reduced susceptibility to PRPs.

Borderline susceptibility to methicillin in Staphylococcus aureus: a new mechanism of resistance?

Staphylococcus aureus strains with borderline levels of susceptibility or resistance to antistaphylococcal penicillinase-resistant penicillins (PRPs) were initially reported as neither heteroresistant nor multiply resistant organisms, producing large amounts of beta-lactamase, and becoming fully susceptible to PRPs in the presence of beta-lactamase inhibitors. This borderline susceptibility or low-level resistance was suggested to be due to beta-lactamase hyperproduction: according to this hypothesis, the staphylococcal beta-lactamase, when hyperproduced, would succeed in partially hydrolyzing methicillin and related PRPs. However, later studies demonstrated that borderline PRP susceptibility cannot be explained soley on this basis, beta-lactamase hyperproduction being neither sufficient nor necessary to determine the borderline phenotype. Intrinsic mechanisms have also been reported to be associated with some borderline PRP susceptible S. aureus strains. The more recent discovery of a PRP-hydrolyzing beta-lactamase (methicillinase) produced, in addition to the conventional penicillinase, by borderline S. aureus strains suggests that this second, more specific beta-lactamase is more likely to be responsible for the borderline phenotype than an increased amount of the penicillinase. The slow kinetics of PRP hydrolysis by methicillinase is consistent with its association with reduced susceptibility rather than true resistance to PRPs. The combined effect of methicillinase plus penicillinase on some common substrates might explain the increased beta-lactamase activity often observed in borderline S. aureus strains.

Cloning and expression of the penicillinase from a borderline methicillin-susceptible Staphylococcus aureus strain in Escherichia coli.

The blaZ gene contained in a single 17.2-kb beta-lactamase plasmid from a borderline methicillin-susceptible Staphylococcus aureus strain (a53) has been cloned in Escherichia coli. A Bluescript II derivative in which the ampicillin resistance gene has been replaced with the chloramphenicol resistance gene was used as a multi-copy vector. One ampicillin-resistant colony was detected among 31 chloramphenicol-resistant transformants selected. This E. coli clone harbored a recombinant plasmid (pAH12) containing two different staphylococcal HindIII inserts (7.0 and 5.3 kb), of which only the former hybridized with a blaZ probe. The clone showed an ampicillin MIC of > 1024 micrograms ml-1, independently of the inoculum size used, and produced large amounts of beta-lactamase, which hydrolyzed nitrocefin and penicillin G but not methicillin of the beta-lactamase substrate, padac. In contrast, S. aureus a53 hydrolyzed all four substrates. The fact that high levels of staphylococcal penicillinase are unable to cause methicillin hydrolysis confirms that penicillinase hyperproduction is unlikely to be the true mechanism responsible for the borderline phenotype. These results also suggest that the two different beta-lactamases (penicillinase and methicillinase) associated with borderline S. aureus strains have a different genetic origin.

Survey of clinical isolates of Staphylococcus aureus for borderline susceptibility to antistaphylococcal penicillins.

On the basis of the MICs of methicillin and oxacillin, 975 clinical isolates of Staphylococcus aureus were categorized as having resistance, borderline susceptibility or full susceptibility to penicillinase-resistant penicillins (PRPs). The borderline phenotype accounted for 122 isolates (12.5%), whereas 562 isolates were fully susceptible and 290 resistant; one remaining isolate had resistance to methicillin and borderline susceptibility to oxacillin. Reductions in the MICs of methicillin and oxacillin in the presence of sulbactam were greater in strains with borderline PRP susceptibility than in fully susceptible or resistant isolates. Over 99% of fully PRP-susceptible strains, 93% with borderline susceptibility and 71% of resistant strains were susceptible to ampicillin/sulbactam. The production of beta-lactamase, assayed in all strains using nitrocefin as substrate, could be detected without prior induction in 729 strains and after induction only in another 156 strains. With only two exceptions, the beta-lactamase negative strains were part of the fully PRP-susceptible group of organisms (88 of 562 isolates). Among the borderline isolates, strong beta-lactamase reactions were encountered with particular frequency, but not in all strains and not exclusively in borderline strains. Although associated with the majority of borderline strains, beta-lactamase hyperproduction thus did not appear to be an essential feature of the borderline phenotype. The results obtained may have implications for laboratory and clinical medicine, also in the light of recent findings suggesting that other mechanisms besides beta-lactamase hyperproduction may account for borderline susceptibility to PRPs.

Evidence for a methicillin-hydrolysing beta-lactamase in Staphylococcus aureus strains with borderline susceptibility to this drug.

A new beta-lactamase that hydrolyses methicillin was found in the membrane fraction of two clinical isolates of Staphylococcus aureus with borderline susceptibility to this drug. 'Methicillinase' activity was detected in renatured sodium dodecyl sulfate polyacrylamide gel electrophoretograms of staphylococcal membrane proteins. The enzyme activity appeared to be inducible and was more easily detected using penicillin G (or methicillin) rather than nitrocefin as substrate. Similar activity was not detected in the membrane fraction of a methicillin-susceptible strain. These results suggest that, in the two borderline susceptible strains, rather than a hyperproduction of the penicillinase a specific methicillin-hydrolysing activity is responsible for the borderline susceptible phenotype.

Haemophilus parainfluenzae causing sexually transmitted urethritis. Report of a case and evidence for a beta-lactamase plasmid mobilizable to Escherichia coli by an Inc-W plasmid.

A multiple antibiotic resistance, beta-lactamase-producing strain of Haemophilus parainfluenzae was isolated from a patient with sexually transmitted urethritis that was contracted in Northwest Africa. The strain was found to harbor a small (3.2 megadaltons) plasmid encoding for beta-lactamase production, which was successfully mobilized to Escherichia coli in triparental mating experiments by means of a broad host-range Inc-W conjugative plasmid. Since H. parainfluenzae is believed to be a source and reservoir for the spread of beta-lactamase plasmids to other bacterial species, such a plasmid mobilization may suggest a new possible means for resistance plasmid dissemination.

Development of in-vitro resistance to glycopeptide antibiotics: assessment in staphylococci of different species.

Forty-two clinical isolates belonging to ten species of staphylococci were studied for their ability to develop single-step resistance, in vitro, to glycopeptide antibiotics. Selection was attempted through separate exposure to four glycopeptides (vancomycin, teicoplanin, and two investigational semisynthetic derivatives of the latter, TD-A3 and CTA-A1) on agar containing 10 mg/l of the test drug. No survivors from any test strain were recovered after exposure to TD-A3 or CTA-A1. After exposure to vancomycin or teicoplanin, surviving clones were only recovered from strains of three species, Staphylococcus aureus, S. epidermidis, and S. haemolyticus. Emergence of resistant clones was easier to observe from strains of S. haemolyticus exposed to teicoplanin. When tested for susceptibility, many survivors exhibited vancomycin and teicoplanin MICs below the drug concentration used for in-vitro selection, probably due to an inoculum effect in the plating procedure. In particular, the vancomycin MICs did not exceed 8 mg/l for S. aureus and S. epidermidis clones, and reached 16 mg/l for some clones from a S. haemolyticus strain. Teicoplanin MICs did not exceed 8 mg/l for S. aureus clones, but reached 64 mg/l for some clones of S. epidermidis, and were particularly high (64 to greater than or equal to 128 mg/l) for most clones of S. haemolyticus. In contrast, against all clones selected from all three species, the MICs of TD-A3 and CTA-A1 did not exceed 2 and 4 mg/l, respectively. Morphological investigations indicated that the colonies of a highly resistant S. haemolyticus clone were smaller and more butyraceous in consistency than those of the parent strain. In transmission and scanning electron microscopy studies, this same S. haemolyticus clone showed a more irregular cell wall than the parent strain.