Daptomycin In Vitro Activity against Methicillin-Resistant Staphylococcus aureus Is Enhanced by d-Cycloserine in a Mechanism Associated with a Decrease in Cell Surface Charge.

The killing activity of daptomycin against an isogenic pair of daptomycin-susceptible and daptomycin-nonsusceptible (DNS) methicillin-resistant Staphylococcus aureus (MRSA) strains was enhanced by the addition of certain cell wall agents at 1× MIC. However, when high inocula of the DNS strain were used, no significant killing was observed in our experiments. Cytochrome c binding assays revealed d-cycloserine as the only agent associated with a reduction in the cell surface charge for both strains at the concentrations used.

Utility of peptide nucleic acid fluorescence in situ hybridization for rapid detection of Acinetobacter spp. and Pseudomonas aeruginosa.

The utility of peptide nucleic acid fluorescence in situ hybridization (PNA FISH) for the detection of Acinetobacter spp. and Pseudomonas aeruginosa was evaluated on broth suspensions and spiked blood cultures of ATCC strains and clinical isolates with select gram-negative rods. After testing 60 clinical isolates, PNA FISH had a sensitivity and specificity of 100% and 100%, respectively, for Acinetobacter spp. and 100% and 95%, respectively, for P. aeruginosa. PNA FISH was able to detect both pathogens simultaneously and directly from spiked blood cultures.

Antimicrobial agents for treatment of serious infections caused by resistant Staphylococcus aureus and enterococci.

As clinicians increasingly contend with infections due to staphylococci or enterococci resistant to, or failing treatment with, traditional antimicrobial agents, understanding the potential roles of older as well as more recently introduced antimicrobial agents becomes important. Older agents, such as clindamycin and trimethoprim-sulfamethoxazole, have been used to treat infections due to community-acquired methicillin-resistant Staphylococcus aureus. Among the licensed agents, quinupristin-dalfopristin, linezolid, daptomycin, and tigecycline are active in vitro against most strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium, but these agents differ in their approved clinical indications. New agents currently under investigation may further expand treatment options.

In vitro activity of an oral streptogramin antimicrobial, XRP2868, against gram-positive bacteria.

The comparative in vitro potency of XRP2868, a new oral semisynthetic streptogramin antibiotic, was evaluated against gram-positive bacteria. XRP2868 inhibited all staphylococci at < or = 1 microg/ml and all non-pneumococcal streptococci at < or = 0.25 microg/ml and was fourfold more potent than quinupristin-dalfopristin against Staphylococcus aureus and Enterococcus faecium.

Safety and efficacy of glycopeptide antibiotics.

It would be difficult to envision the practice of infectious diseases over the past 20 years without the availability of the glycopeptide antibiotics. The two agents currently in clinical use, vancomycin and teicoplanin, have proven remarkably versatile in many common applications. Several attributes of these agents account for this favourable profile: (i) their broad spectrum of activity against Gram-positive bacteria, including strains resistant to many other antimicrobials; (ii) their favourable pharmacokinetic properties that allow the once- or twice-daily dosing regimens that have made out-of-hospital therapy possible; and (iii) their generally good safety profiles which, along with their structural dissimilarity to beta-lactam and other antimicrobials, permits their use in many patients who are intolerant of other antibiotic regimens. It is not entirely surprising, therefore, that despite more than 40 years of clinical use and the interim appearance of bacterial strains resistant to this drug class, there remains continued interest in the development of newer members of the glycopeptide antibiotic class. This paper is intended to provide a global overview of the efficacy and safety of glycopeptide antibiotics currently in use, as background to understanding the need for and potential roles of new agents of this class.

In vitro activity of the new oxazolidinone AZD2563 against Enterococci.

The activity of a new oxazolidinone antimicrobial, AZD2563, was assessed against >500 clinical isolates of enterococci representing six species. All isolates, including those resistant to other antibiotic classes, were inhibited by AZD2563 at concentrations

Prevalence of the fsr locus in Enterococcus faecalis infections.

The fsr locus of Enterococcus faecalis confers virulence in animal models. A retrospective analysis of fsr prevalence in diverse E. faecalis clinical isolates demonstrated fsr in all endocarditis isolates versus 53% of stool isolates (P = 0.005). This supports a role for fsr-mediated virulence in the pathogenesis of enterococcal infections in humans.

Antimicrobial activity of quinupristin-dalfopristin combined with other antibiotics against vancomycin-resistant enterococci.

Interactions between quinupristin-dalfopristin and six other antimicrobials were examined by checkerboard arrays against 50 clinical isolates of vancomycin-resistant Enterococcus faecium selected to represent a range of susceptibilities to individual agents. Unequivocal synergistic or antagonistic interactions at clinically relevant concentrations were infrequently encountered when the streptogramin was combined with chloramphenicol, ampicillin, imipenem, vancomycin, or teicoplanin. Combinations with doxycycline resulted in synergistic inhibition in 36% of checkerboards. Against 10 strains of Enterococcus faecalis, synergistic interactions were found when quinupristin-dalfopristin was combined with doxycycline (four strains), either glycopeptide (three strains), or ampicillin (two strains). Combination with quinupristin-dalfopristin increased the ampicillin MIC from 1 to 4 microg/ml for one strain. For 10 strains of E. faecium, interactions were also assessed by time-kill methods using concentrations of the agents attainable in human serum. Most of these antimicrobials augmented killing by quinupristin-dalfopristin to a minor degree. Against 2 of the 12 strains in this collection that were not highly resistant to gentamicin, the combination of quinupristin-dalfopristin (2 microg/ml) plus gentamicin (5 microg/ml) resulted in killing approaching 3 log(10) CFU/ml. With the exception of doxycycline, inhibitory interactions between quinupristin-dalfopristin and other agents tested against vancomycin-resistant strains of E. faecium were uncommon at clinically relevant concentrations.

Methicillin-resistant Staphylococcus aureus: comparison of susceptibility testing methods and analysis of mecA-positive susceptible strains.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections. Phenotypic heterogeneous drug resistance (heteroresistance) to antistaphylococcal beta-lactams affects the results of susceptibility testing. The present study compared the MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) for detection of PBP 2a with agar dilution, the VITEK-1 and VITEK-2 systems (bioMérieux, St. Louis, Mo.), and the oxacillin agar screen test for detection of MRSA, with PCR for the mecA gene used as the "gold standard" assay. Analysis of 107 methicillin-susceptible S. aureus (MSSA) isolates and 203 MRSA isolates revealed that the MRSA-Screen latex agglutination test is superior to any single phenotype-based susceptibility testing method, with a sensitivity of 100% and a specificity of 99.1%. Only one isolate that lacked mecA was weakly positive by the MRSA-Screen latex agglutination test. This isolate was phenotypically susceptible to oxacillin and did not contain the mecA gene by Southern blot hybridization. The oxacillin agar screen test, the VITEK-1 system, the VITEK-2 system, and agar dilution showed sensitivities of 99.0, 99.0, 99.5, and 99%, respectively, and specificities of 98.1, 100, 97.2, and 100%, respectively. The differences in sensitivity or specificity were not statistically significant. Oxacillin bactericidal assays showed that mecA- and PBP 2a-positive S. aureus isolates that are susceptible to antistaphylococcal beta-lactams by conventional methods are functionally resistant to oxacillin. We conclude that the accuracy of the MRSA-Screen latex agglutination method for detection of PBP 2a approaches the accuracy of PCR and is more accurate than any susceptibility testing method used alone for the detection of MRSA.

Prolonged colonization with vancomycin-resistant Enterococcus faecium in long-term care patients and the significance of "clearance".

Little is known about the persistence of colonization with vancomycin-resistant Enterococcus faecium (VRE) in the nononcologic, non-intensive care unit patient. We studied all patients who had VRE isolated on > or =2 occasions of > 1 year apart (Study A) and those who had been "cleared" of VRE colonization after 3 negative stool cultures (Study B). Twelve patients had stored VRE isolates recovered > 1 year apart (Study A), and 58% of paired isolates were genotypically related according to pulsed field gel electrophoresis patterns. In Study B, stool samples were obtained weekly from 21 "cleared" patients for 5 weeks, which revealed that 24% were VRE positive. For these culture-positive patients, 72% of the cultures failed to detect VRE. Recent antibiotic use was significantly more common in the culture-positive patients, as compared with culture-negative patients (P=.003). Colonization with VRE may persist for years, even if the results of intercurrent surveillance stool and index site cultures are negative. Cultures for detection of VRE in stool samples obtained from patients declared "cleared" are insensitive.

Risk factors for emergence of resistance to broad-spectrum cephalosporins among Enterobacter spp.

Among 477 patients with susceptible Enterobacter spp., 49 subsequently harbored third-generation cephalosporin-resistant Enterobacter spp. Broad-spectrum cephalosporins were independent risk factors for resistance (relative risk [OR] = 2.3, P = 0.01); quinolone therapy was protective (OR = 0.4, P = 0.03). There were trends toward decreased risk for resistance among patients receiving broad-spectrum cephalosporins and either aminoglycosides or imipenem. Of the patients receiving broad-spectrum cephalosporins, 19% developed resistance.

Linezolid resistance in a clinical isolate of Staphylococcus aureus.

The new oxazolidinone antimicrobial, linezolid, has been approved for the treatment of infections caused by various gram-positive bacteria, including meticillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Although instances of linezolid resistance in VRE have been reported, resistance has not been encountered among clinical isolates of S aureus. We have characterised an MRSA isolate resistant to linezolid that was recovered from a patient treated with this agent for dialysis-associated peritonitis.

Restoration of vancomycin susceptibility in Enterococcus faecalis by antiresistance determinant gene transfer.

We assessed the ability of gene transfer to reverse vancomycin resistance in class A (VanA) glycopeptide-resistant Enterococcus faecalis. Recombinant shuttle vectors containing a vanH promoter-vanA antisense gene cassette fully restored vancomycin susceptibility through a combined transcriptional activator binding domain decoy and inducible vanA antisense RNA effect.

Quinolones and false-positive urine screening for opiates by immunoassay technology.

CONTEXT: Millions of assays are performed each year to monitor for substance abuse in various settings. When common medications cross-react with frequently used testing assays, false-positive results can lead to invalid conclusions. OBJECTIVE: To evaluate cross-reactivity of quinolone antimicrobials in common opiate screening assays and to assess the in vivo implications of this phenomenon. DESIGN, SETTING, AND PARTICIPANTS: The reactivity of 13 quinolones (levofloxacin, ofloxacin, pefloxacin, enoxacin, moxifloxacin, gatifloxacin, trovafloxacin, sparfloxacin, lomefloxacin, ciprofloxacin, clinafloxacin, norfloxacin, and nalidixic acid) was tested in 5 commercial opiate screening assays from September 1998 to March 1999. In 6 healthy volunteers, we confirmed the cross-reactivity of levofloxacin or ofloxacin with these opiate screening assays. MAIN OUTCOME MEASURE: Opiate assay activity (threshold for positive result, 300 ng/mL of morphine). RESULTS: Nine of the quinolones caused assay results above the threshold for a positive result in at least 1 of the assays. Four of the assay systems caused false-positive results for at least 1 quinolone. Eleven of the 13 compounds caused some opiate activity by at least 1 assay system. At least 1 compound caused opiate assay activity in all 5 assay systems. Levofloxacin, oflaxacin, and perfloxacin were most likely to lead to a false-positive opiate result. Positive results were obtained in urine from all 6 volunteers. CONCLUSION: Greater attention to the cross-reactivity of quinolones with immunoassays for opiates is needed to minimize the potential for invalid test interpretation.

In vivo activity of evernimicin (SCH 27899) against methicillin-resistant Staphylococcus aureus in experimental infective endocarditis.

Currently, there exist few satisfactory alternatives to vancomycin for therapy of serious methicillin-resistant Staphylococcus aureus (MRSA) infections. We employed a rat model of aortic valve endocarditis to assess the potential efficacy of evernimicin (SCH 27899) compared with vancomycin against infection with a strain susceptible to both agents (MICs of 0.25 and 0.50 microg/ml, respectively). Infected animals were assigned to one of three groups: controls (no treatment), evernimicin at 60 mg/kg of body weight by intravenous (i.v.) infusion once daily, or vancomycin at 150 mg/kg of body weight per day by continuous i.v. infusion. Therapy was administered for 5.5 days. At the start of therapy, colony counts in vegetations were 6.63 +/- 0.44 log(10) CFU/g. In both treatment groups, bacterial density within vegetations was significantly reduced in comparison with control animals that had not been treated. Final colony counts were as follows (mean +/- standard deviation): controls, 10.12 +/- 1.51 log(10) CFU/g of vegetation; evernimicin, 7.22 +/- 2.91 log(10) CFU/g of vegetation; vancomycin, 5.65 +/- 1.76 log(10) CFU/g of vegetation. The difference between the evernimicin and vancomycin groups was not significant. These results confirmed the bacteriostatic activity of evernimicin in vivo in an experimental model of severe MRSA infection.

Clinical isolation and resistance patterns of and superinfection with 10 nosocomial pathogens after treatment with ceftriaxone versus ampicillin-sulbactam.

Isolation of pathogens from clinical cultures and their resistance patterns may be altered by antecedent antibiotic treatment. The objective of this study was to assess the influence of treatment with ceftriaxone versus that with ampicillin-sulbactam on recovery and superinfections with 10 nosocomial pathogens. The study was designed as a historical cohort study, using a propensity score to adjust for confounding by indication and multivariate survival analyses to adjust for other confounding. Two thousand four hundred forty-five patients were treated with ampicillin-sulbactam, and 1, 308 were treated with ceftriaxone. The study analyzed two outcomes: (i) recovery of pathogens from clinical cultures and (ii) microbiologically documented infections. Data were obtained from administrative, pharmacy, clinical, and laboratory databases and by chart extraction. Following treatment, new isolation of at least 1 of the 10 target pathogens occurred for 244 patients. After adjustment, more infections occurred in the ampicillin-sulbactam group (hazard ratio [HR], 1.55; P = 0.009). This was observed with all gram-negative rods combined (HR, 3.6; P < 0.001) and with each genus of the family Enterobacteriaceae. No differences in isolation of gram-positive bacteria were evident (P = 0.33). Microbiologically documented superinfections occurred in 172 patients and were less frequent in the ceftriaxone group (3.8% versus 5%; HR, 1.6; P = 0. 015). All the Escherichia coli and Klebsiella spp. isolates were susceptible to ceftriaxone, but half were resistant to ampicillin-sulbactam. The prevalence of oxacillin resistance among Staphylococcus aureus isolates was higher in the ceftriaxone group (63% versus 31%; odds ratio, 3.8; P = 0.08). Differences in the rates of superinfections and the likely causative organisms following treatment with ceftriaxone or ampicillin-sulbactam were evident. This may guide clinicians in empirical choices of antibiotics to treat superinfection.

Diversity of domain V of 23S rRNA gene sequence in different Enterococcus species.

The highly conserved central loop of domain V of 23S RNA (nucleotides 2042 to 2628; Escherichia coli numbering) is implicated in peptidyltransferase activity and represents one of the target sites for macrolide, lincosamide, and streptogramin B antibiotics. DNA encoding domain V (590 bp) of several species of Enterococcus was amplified by PCR. Twenty enterococcal isolates were tested, including Enterococcus faecium (six isolates), Enterococcus faecalis, Enterococcus avium, Enterococcus durans, Enterococcus gallinarum, Enterococcus casseliflavus (two isolates of each), and Enterococcus raffinosus, Enterococcus mundtii, Enterococcus malodoratus, and Enterococcus hirae (one isolate of each). For all isolates, species identification by biochemical testing was corroborated by 16S rRNA gene sequencing. The sequence of domain V of the 23S rRNA gene from E. faecium and E. faecalis differed from those of all other enterococci. The domain V sequences of E. durans and E. hirae were identical. This was also true for E. gallinarum and E. casseliflavus. E. avium differed from E. casseliflavus by 23 bases, from E. durans by 16 bases, and from E. malodoratus by 2 bases. E. avium differed from E. raffinosus by one base. Despite the fact that domain V is considered to be highly conserved, substantial differences were identified between several enterococcal species.

SME-type carbapenem-hydrolyzing class A beta-lactamases from geographically diverse Serratia marcescens strains.

Three sets of carbapenem-resistant Serratia marcescens isolates have been identified in the United States: 1 isolate in Minnesota in 1985 (before approval of carbapenems for clinical use), 5 isolates in Los Angeles (University of California at Los Angeles [UCLA]) in 1992, and 19 isolates in Boston from 1994 to 1999. All isolates tested produced two beta-lactamases, an AmpC-type enzyme with pI values of 8.6 to 9.0 and one with a pI value of approximately 9.5. The enzyme with the higher pI in each strain hydrolyzed carbapenems and was not inhibited by EDTA, similar to the chromosomal class A SME-1 beta-lactamase isolated from the 1982 London strain S. marcescens S6. The genes encoding the carbapenem-hydrolyzing enzymes were cloned in Escherichia coli and sequenced. The enzyme from the Minnesota isolate had an amino acid sequence identical to that of SME-1. The isolates from Boston and UCLA produced SME-2, an enzyme with a single amino acid change relative to SME-1, a substitution from valine to glutamine at position 207. Purified SME enzymes from the U. S. isolates had beta-lactam hydrolysis profiles similar to that of the London SME-1 enzyme. Pulsed-field gel electrophoresis analysis revealed that the isolates showed some similarity but differed by at least three genetic events. In conclusion, a family of rare class A carbapenem-hydrolyzing beta-lactamases first described in London has now been identified in S. marcescens isolates across the United States.

In vivo activities of evernimicin (SCH 27899) against vancomycin-susceptible and vancomycin-resistant enterococci in experimental endocarditis.

To assess the potential efficacy of evernimicin (SCH 27899) against serious enterococcal infections, we used a rat model of aortic valve endocarditis established with either a vancomycin-susceptible Enterococcus faecalis or a vancomycin-resistant Enterococcus faecium strain. Animals infected with either one of the test strains were assigned to receive no treatment (controls) or 5-day therapy with one of the following regimens: evernimicin 60-mg/kg of body weight intravenous (i.v.) bolus once daily, 60-mg/kg i.v. bolus twice daily (b.i.d.), 60 mg/kg/day i.v. by continuous infusion, or 120 mg/kg/day i.v. by continuous infusion. These regimens were compared with vancomycin at 150 mg/kg/day. In animals infected with E. faecalis, evernimicin at 120 mg/kg/day by continuous infusion significantly reduced bacterial counts in vegetations (final density, 5.75+/-3.38 log(10) CFU/g) compared with controls (8.51+/-1.11 log(10) CFU/g). In animals infected with 0.5 ml of an 8 x 10(7)-CFU/ml inoculum of the vancomycin-resistant E. faecium, both 60-mg/kg bolus once a day and b.i.d. dose regimens of evernimicin were very effective (viable counts, 3.45+/-1.44 and 3.81+/-1.98 log(10) CFU/g, respectively). Vancomycin was unexpectedly active against infections induced with that inoculum. In animals infected with a 10(9)-CFU/ml inoculum of the vancomycin-resistant E. faecium, the evernimicin 60-mg/kg i.v. bolus b.i.d. reduced viable counts in vegetations compared with controls (6.27+/-1.63 versus 8.34+/-0.91 log(10) CFU/g; P<0.05), whereas vancomycin was ineffective. Although resistant colonies could be selected in vitro, we were not able to identify evernimicin-resistant clones from cardiac vegetations. An unexplained observation from these experiments was the great variability in final bacterial densities within cardiac vegetations from animals in each of the evernimicin treatment groups.