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Comparative efficacies of liposomal amikacin (MiKasome) plus oxacillin versus conventional amikacin plus oxacillin in experimental endocarditis induced by Staphylococcus aureus: microbiological and echocardiographic analyses.

Optimal treatment strategies for serious infections caused by Staphylococcus aureus have not been fully characterized. The combination of a beta-lactam plus an aminoglycoside can act synergistically against S. aureus in vitro and in vivo. MiKasome, a new liposome-encapsulated formulation of conventional amikacin, significantly prolongs serum half-life (t1/2) and increases the area under the concentration-time curve (AUC) compared to free amikacin. Microbiologic efficacy and left ventricular function, as assessed by echocardiography, were compared in animals administered either oxacillin alone or oxacillin in combination with conventional amikacin or MiKasome in a rabbit model of experimental endocarditis due to S. aureus. In vitro, oxacillin, combined with either free amikacin or MiKasome, prevented the bacterial regrowth observed with aminoglycosides alone at 24 h of incubation. Rabbits with S. aureus endocarditis were treated with either oxacillin alone (50 mg/kg, given intramuscularly three times daily), oxacillin plus daily amikacin (27 mg/kg, given intravenously twice daily), or oxacillin plus intermittent MiKasome (160 mg/kg, given intravenously, a single dose on days 1 and 4). The oxacillin-alone dosage represents a subtherapeutic regimen against the infecting strain in the endocarditis model (L. Hirano and A. S. Bayer, Antimicrob. Agents Chemother. 35:685-690, 1991), thus allowing recognition of any enhanced bactericidal effects between oxacillin and either aminoglycoside formulation. Treatment was administered for either 3 or 6 days, and animals were sacrificed after each of these time points or at 5 days after a 6-day treatment course (to evaluate for posttherapy relapse). Left ventricular function was analyzed by utilizing serial transthoracic echocardiography during treatment and posttherapy by measurement of left ventricular fractional shortening. At all sacrifice times, both combination regimens significantly reduced S. aureus vegetation counts versus control counts (P < 0.05). In contrast, oxacillin alone did not significantly reduce S. aureus vegetation counts after 3 days of therapy. Furthermore, at this time point, the two combinations were significantly more effective than oxacillin alone (P < 0.05). All three regimens were effective in significantly decreasing bacterial counts in the myocardium during and after therapy compared to controls (P < 0.05). In kidney and spleen abscesses, all regimens significantly reduced bacterial counts during therapy (P < 0.0001); however, only the combination regimens prevented bacteriologic relapse in these organs posttherapy. By echocardiographic analysis, both combination regimens yielded a significant physiological benefit by maintaining normal left ventricular function during treatment and posttherapy compared with oxacillin alone (P < 0.001). These results suggest that the use of intermittent MiKasome (similar to daily conventional amikacin) enhances the in vivo bactericidal effects of oxacillin in a severe S. aureus infection model and preserves selected physiological functions in target end organs.

Amikacin↗

Evaluation of laboratory methods for the classification of oxacillin-resistant and oxacillin-susceptible Staphylococcus aureus.

The purpose of this study was to examine the efficacy of the oxacillin disk diffusion test and the methicillin and oxacillin agar screen tests as predictors of oxacillin resistance as defined by the reference broth microdilution method. A total of 444 clinical isolates of Staphylococcus aureus collected from individual patients over a four-year period were tested by (1) the oxacillin disk diffusion test, with particular attention to the presence of fine growth of a resistant subpopulation within the zone of inhibition (Ox Grow interpretive criteria); (2) the agar screen method using agar plates with 4% NaCl and either 6 mg/L oxacillin or 10 mg/L methicillin; and (3) the oxacillin and methicillin broth microdilution test methods with 2% NaCl supplementation. Overall, 62 (14%) isolates were resistant and 382 (86%) isolates were susceptible to oxacillin with the use of the reference broth microdilution system. The results indicate that the disk diffusion test with the use of the Ox Grow criteria had a high sensitivity (94%) and negative predictive value (98%) but a low specificity (67%) and positive predictive value (32%) when compared with the reference broth dilution test. Similarly, the agar screen tests had a high sensitivity (95-97%) and negative predictive values (99%) but low specificity (64-74%) and positive predictive values (30-37%). These data indicate that the agar screen tests and the oxacillin disk test with the use of the Ox Grow interpretive criteria may be useful as screening tests for detecting resistance to the penicillinase-resistant penicillins but that all resistant isolates should be confirmed by the reference broth dilution method because of the large number of false-resistant screening test results.

Bacteriological Techniques↗

Genetics of oxacillin resistance in clinical isolates of Streptococcus pneumoniae that are oxacillin resistant and penicillin susceptible.

It has recently been reported that penicillin-sensitive pneumococci may exhibit reduced susceptibility to oxacillin, resulting in their misclassification as being penicillin resistant by oxacillin disk testing. Intermediate oxacillin resistance (MIC, 1.0 microgram/ml) in three of these apparently unrelated penicillin-susceptible clinical isolates of Streptococcus pneumoniae isolated in the United Kingdom and in four Spanish isolates was shown to be solely due to the acquisition of a gene encoding an altered penicillin-binding protein (PBP), PBP2X. PBP2X genes cloned from typical penicillin-resistant isolates of S. pneumoniae that possessed high-level oxacillin resistance were shown to be able to transform susceptible isolates of S. pneumoniae to intermediate oxacillin resistance. In all instances, the intermediately oxacillin-resistant PBP2X transformants retained susceptibility to penicillin (MIC, 0.06 microgram/ml). Under appropriate selective pressure, the acquisition of a low-affinity PBP2X by penicillin-susceptible pneumococci could result in an increasing number of false positives for penicillin resistance among isolates of S. pneumoniae screened with oxacillin. Additionally, these intermediately oxacillin-resistant isolates showed reduced susceptibility to cefotaxime, an agent likely to be prescribed in place of penicillin for the treatment of serious infections due to these apparently penicillin-resistant organisms.

Bacterial Proteins↗

Selection of oxacillin resistance in oxacillin-sensitive Staphylococcus aureus through exposure to ciprofloxacin but not piperacillin/tazobactam.

Ten oxacillin-sensitive Staphylococcus aureus strains were grown on agar containing four times their ciprofloxacin MIC to determine if exposure to ciprofloxacin would increase their resistance to oxacillin. All strains grew on the ciprofloxacin-containing agar and subsequently grew on oxacillin-salt agar. The geometric mean MICs for oxacillin increased one- to sixteen-fold and remained elevated after ten passages on antibiotic-free agar. The mecA gene was not detected in any strain. There was no increase in oxacillin MICs when the bacteria were passaged on agar containing four times their MIC of piperacillin/tazobactam. Exposure of oxacillin-sensitive strains of Staphylococcus aureus to ciprofloxacin may increase their MICs to oxacillin.

Anti-Infective Agents↗

Coagulase-negative staphylococci: comparison of phenotypic and genotypic oxacillin susceptibility tests and evaluation of the agar screening test by using different concentrations of oxacillin.

This study evaluated the oxacillin susceptibilities of 152 coagulase-negative staphylococcal (CoNS) strains of 12 species by disk diffusion; agar dilution; E-test; the slide latex agglutination test (Slidex MRSA Detection test; bioMérieux S/A, Paris, France); the agar screening test with 1, 2, 4, or 6 microg of oxacillin per ml and incubation for 24 or 48 h; and detection of the mecA gene by PCR. The results revealed that the agar screening test with 4 micro g of oxacillin per ml and incubation for 48 h was superior to any single phenotype-based susceptibility assay, presenting a sensitivity and a specificity of 100% each. For the different methods evaluated, the sensitivities and specificities were as follows: for disk diffusion, 94.2 and 91.8%, respectively; for the agar dilution test 100 and 73.5%, respectively; for E-test, 100 and 71.4%, respectively; and for the slide latex agglutination test, 97.1 and 98%, respectively. A good correlation was observed between oxacillin susceptibility testing results and PCR results for Staphylococcus epidermidis, S. haemolyticus, S. hominis subsp. hominis, and all mecA-positive strains. However, at least 60% of the mecA-negative isolates of the species S. saprophyticus, S. cohnii subsp. urealyticum, S. lugdunensis, and S. sciuri were erroneously classified as oxacillin resistant by the agar dilution test. Conversely, the slide latex agglutination test presented a high sensitivity (97.1%) and a high specificity (98%) for all CoNS species. Our results demonstrated the accuracy of the agar screening test with 4 micro g of oxacillin per ml and incubation for 48 h and the slide latex agglutination test for the appropriate detection of the oxacillin susceptibilities of CoNS isolates. Both assays are technically simple and can be easier to perform in routine laboratories than PCR.

Agar↗

The utility of non-beta-lactam antimicrobial MICs as markers to distinguish oxacillin-resistant from oxacillin-susceptible strains of Staphylococcus epidermidis.

Among 6,068 strains of Staphylococcus epidermidis, 75.5% were oxacillin-resistant. Oxacillin-susceptible strains were more frequently susceptible to erythromycin, clindamycin, ciprofloxacin, trimethoprim/sulfamethoxazole, gentamicin, and tetracycline than oxacillin-resistant strains. With the exception of erythromycin, non-beta-lactam MICs were less discriminatory for identifying oxacillin-resistant strains with oxacillin MICs < or = 2 micrograms/ml than for those with oxacillin MICs > or = 4 micrograms/ml.

Anti-Bacterial Agents↗

Relationship between cefamandole and cefuroxime activity against oxacillin-resistant Staphylococcus epidermidis and oxacillin resistance phenotype.

The activity of cefamandole and cefuroxime against oxacillin-resistant staphylococcus epidermidis was studied in vitro to determine whether there was any relationship between oxacillin resistance phenotypes and cephalosporin activity. Oxacillin resistance phenotypes were determined by efficiency-of-plating studies on Mueller-Hinton agar containing oxacillin, with and without NaCl, and incubated at 30 and 35 degrees C. On the basis of MIC and MBC determinations, cefamandole was more active than cefuroxime against oxacillin-resistant S. epidermidis. Although temperature had minimal effect on the activity of either cefamandole or cefuroxime, NaCl significantly decreased the activity of cefuroxime but not of cefamandole. Neither cephalosporin consistently produced greater than or equal to 99.9% bactericidal activity within 24 h in timed killing-curve studies. No consistent relationship was observed between cefamandole or cefuroxime activity and oxacillin resistance phenotype.

Cefamandole↗

Branched-DNA assay for detection of the mecA gene in oxacillin-resistant and oxacillin-sensitive staphylococci.

The identification of methicillin-resistant staphylococcus isolates in the clinical laboratory has typically been performed by using methods that detect phenotypic expression of resistance determinants. However, these methods may be difficult to interpret and some isolates do not express resistance until selective pressure is administered. Assays that detect genetic determinants are not subject to these limitations and have been effective in distinguishing isolates that are capable of expressing the resistance phenotype. In this study, a novel branched-DNA (bDNA) hybridization assay was used to test for the mecA gene in 416 clinical staphylococcal isolates. The results were compared with those obtained by a PCR-based assay and oxacillin disk diffusion. For 155 Staphylococcus aureus and 261 coagulase-negative Staphylococcus isolates, the bDNA assay and PCR results were 100% concordant. Among the S. aureus isolates, 20 were MecA+ and 135 were MecA-. For the coagulase-negative staphylococci, 150 were MecA+ and 111 were MecA-. The results from the genotypic detection methods were compared with those obtained by oxacillin disk diffusion. No discrepancies were detected among the S. aureus isolates; however, 10 coagulase-negative isolates were MecA+ but oxacillin sensitive and 1 isolate was MecA- but oxacillin resistant. Oxacillin resistance was induced in 6 of the 10 MecA+ isolates previously classified as oxacillin sensitive. These results suggest that the bDNA method described here is a sensitive and efficient method for detection of methicillin resistance in staphylococci and that genetic detection methods may be useful for detection of potential methicillin resistance in the clinical laboratory.

DNA Primers↗

Antistaphylococcal activity of amoxicillin and ticarcillin when combined with clavulanic acid. Evaluation of oxacillin-resistant and oxacillin-susceptible isolates.

The beta-lactamase inhibitor, clavulanic acid, was combined with amoxicillin and with ticarcillin for in vitro studies with 586 staphylococci: 97 stock cultures of oxacillin-resistant strains recovered before 1982, and 489 blood or wound isolates collected from 40 separate medical centers during 1987-1988 (300 were oxacillin resistant). Over 92% of the staphylococci produced beta-lactamase enzymes and were thus resistant to both penicillins. However, with the addition of clavulanic acid, oxacillin-susceptible strains were rendered susceptible to low concentrations of amoxicillin and ticarcillin. Staphylococcus aureus strains with borderline or partial borderline resistance to penicillinase-resistant penicillins occurred infrequently (72 of 325 S. aureus isolates). Those strains were susceptible to both clavulanic acid combinations, because their methicillin resistance is thought to be due to an excess beta-lactamase production. Strains with chromosomally mediated intrinsic heteroresistance were relatively resistant to both drug combinations. Minimal inhibitory concentration (MIC) breakpoints that best separated those heteroresistant strains from oxacillin-susceptible isolates were as follows: amoxicillin/clavulanic acid, less than or equal to 2.0/1.0 micrograms/ml for susceptible; and ticarcillin/clavulanic acid, less than or equal to 4.0/2.0 micrograms/ml for susceptible. When the broth was supplemented by 2% NaCl, MICs for both drug combinations were increased by less than one doubling dilution. Although oxacillin and methicillin broth microdilution tests were more reliable when 2% NaCl was added, tests with the two drug combinations were only minimally improved by adding 2% NaCl to the broth medium.

Amoxicillin↗

Imipenem and meropenem activity against mecA-positive homogeneously and heterogeneously oxacillin-resistant and mecA-negative oxacillin-borderline-susceptible staphylococci.

Microbroth dilution and disk-diffusion testing of imipenem and meropenem was performed at 35 and 30 degrees C against 61 phenotypic expression class 3,4 and 9 phenotypic expression class 1,2 oxacillin-resistant isolates of Staphylococcus aureus (ORSA), 51 oxacillin-borderline-susceptible isolates of S. aureus (BORSA), and 37 phenotypic expression class 3,4 and 9 phenotypic expression class 1,2 isolates of Staphylococcus epidermidis (ORSE). Imipenem MIC ranges at 35 degree C were 0.6 to > 64 micrograms/ml for class 3,4 ORSA, 0.03 to 0.25 micrograms/ml for class 1,2 ORSA, 0.015 to 0.12 micrograms/ml for BORSA, 0.03 to 64 micrograms/ml for class 3,4 ORSE, and 0.12 to 8 micrograms/ml for class 1,2 ORSE. Corresponding values for meropenem were 0.5 to 64 micrograms/ml, 0.12 to 4 micrograms/ml, 0.06 to 1 microgram/ml, 0.5 to 64 micrograms/ml, and 1 to 8 microgram/ml. MIC ranges at 30 degrees C did not differ by more than 1 log2 dilution from those at 35 degrees C. After 24 h incubation of disk-diffusion tests at 35 degrees C, 44% of class 3,4 and 100% of class 1,2 ORSA isolates were imipenem-susceptible; after an additional 24 h at 25 degrees C, 39 and 100% of these isolates, respectively, remained susceptible to imipenem. Similar values were obtained with 24 h incubation at 30 degrees C followed by 24 h at 25 degrees C. All BORSA isolates were susceptible to imipenem. Of the ORSE isolates, 22 and 78% of isolates in classes 3,4 and 1,2, respectively, were susceptible at 24 h with little change after an additional 24 h at 25 degrees C. Similar trends were observed with meropenem. In parallel disk-diffusion studies with oxacillin, false-susceptibility rates of 5% of class 3,4 and 44% class 1,2 ORSA isolates after 24 h of incubation at 35 degrees C were reduced to 3 and 0%, respectively, after an additional 24 h of incubation at 25 degrees C. Imipenem- and meropenem-resistant subpopulations of oxacillin-resistant staphylococci did not seem to be detected by altered susceptibility testing conditions.

Anti-Bacterial Agents↗

Mupirocin resistance among consecutive isolates of oxacillin-resistant and borderline oxacillin-resistant Staphylococcus aureus at a university hospital.

Mupirocin resistance was determined in consecutive oxacillin-resistant and borderline oxacillin-resistant Staphylococcus aureus clinical isolates collected over 14 months at a university hospital during 1991 and 1992. Twenty of 86 (23%) oxacillin-resistant and borderline oxacillin-resistant S. aureus isolates were mupirocin resistant; 80% were high-level resistant. Prior mupirocin use was a significant risk factor (relative risk, 6.08; 95% confidence interval, 3.7 to 9.99). Seven of 20 resistant isolates were distinct strains, as determined by pulsed-field gel electrophoresis typing. Two instances of clonal dissemination of a single strain occurred, but several other distinct mupirocin-resistant strains were documented. Mupirocin resistance was unexpectedly common among these isolates.

DNA, Bacterial↗

[Spectrophotometric method of determining the oxacillin in oxacillin preparations].

The early described procedure for quantitative determination of oxacillin with the spectrophotometric method was used for the assay oxacillin tablets, capsules and injection preparations. It was shown that the excipients used in tablets and capsules did not interfere with oxacillin determination. The reproducibility of the method is +/- 1.5--2 per cent. The results of oxacillin determination with the new method and the currently used methods were compared.

Capsules↗

In vitro activities of ampicillin-sulbactam and cefoperazone-sulbactam against oxacillin-susceptible and oxacillin-resistant staphylococci.

Ampicillin-sulbactam and cefoperazone-sulbactam were tested against staphylococci that were collected from 40 different medical centers throughout the United States. Oxacillin-resistant strains were resistant to both drug combinations, but oxacillin-susceptible strains were uniformly susceptible. The latter included strains with borderline susceptibility to oxacillin and methicillin.

Ampicillin↗

Optimal inoculation methods and quality control for the NCCLS oxacillin agar screen test for detection of oxacillin resistance in Staphylococcus aureus.

To define more precisely the inoculation methods to be used in the oxacillin screen test for Staphylococcus aureus, we tested agar screen plates prepared in house with 6 microg of oxacillin/ml and 4% NaCl using the four different inoculation methods that would most likely be used by clinical laboratories. The organisms selected for testing were 19 heteroresistant mecA-producing strains and 41 non-mecA-producing strains for which oxacillin MICs were near the susceptible breakpoint. The inoculation method that was preferred by all four readers and that resulted in the best combination of sensitivity and specificity was a 1-microl loopful of a 0.5 McFarland suspension. A second objective of the study was to then use this method to inoculate plates from five different manufacturers of commercially prepared media. Although all commercial media performed with acceptable sensitivity compared to the reference lot, one of the commercial lots demonstrated a lack of specificity. Those lots of oxacillin screen medium that fail to grow heteroresistant strains can be detected by using S. aureus ATCC 43300 as a positive control in the test and by using transmitted light to carefully examine the plates for any growth. However, lack of specificity with commercial lots may be difficult to detect using any of the current quality control organisms.

Bacteriological Techniques↗

Vancomycin treatment of bacteremia caused by oxacillin-resistant Staphylococcus aureus: comparison with beta-lactam antibiotic treatment of bacteremia caused by oxacillin-sensitive Staphylococcus aureus.

The epidemiology and therapy of 29 episodes of bacteremia caused by oxacillin- and aminoglycoside-resistant Staphylococcus aureus (OARSA) were compared with 29 episodes of bacteremia due to oxacillin-sensitive S. aureus (OSSA) that occurred during a 36-month period. Patients with bacteremia due to OSSA were younger (P less than 0.05) and were admitted more frequently with acute traumatic injury (P less than 0.01). The overall survival rate one month after persistent bacteremia was 74% for patients with OARSA bacteremia treated with vancomycin compared with 70% for patients with OSSA bacteremia treated with a beta-lactam antibiotic. The results indicate that vancomycin is an effective antibiotic for the treatment of bacteremia caused by OARSA and suggest that its effectiveness is comparable to that of beta-lactam antibiotic treatment of bacteremia due to OSSA.

Anti-Bacterial Agents↗

Phage pattern-specific oxacillin-resistant and borderline oxacillin-resistant Staphylococcus aureus in U.S. hospitals: epidemiological significance.

For a 13-year period (1978 through 1990), oxacillin-resistant (MIC, greater than 4 micrograms/ml) Staphylococcus aureus (ORSA) strains were collected from Clinical Center (National Institutes of Health) patients and patients from five other U.S. hospitals. From Clinical Center patients, 251 of 253 isolates (99%) were bacteriophage typed as phage group III. Five other hospitals contributed 203 ORSA strains, of which 188 (93%) were group III. The group III ORSA strains predominantly included a characteristic core pattern of phages, 7/47/53/54/75/77. For the low-level (borderline) oxacillin-resistant strains (MIC, 2 to 4 micrograms/ml), amoxicillin-clavulanic acid combination (Augmentin) testing disclosed 62 hyper-beta-lactamase producers, of which 59 (95%) were of a separate, distinct S. aureus strain, with the phage pattern 92/94/96/292/D-11 (group V). Thus, ORSA and hyper-beta-lactamase producing S. aureus are distinct epidemic strains.

Bacteriophage Typing↗

New latex reagent using monoclonal antibodies to capsular polysaccharide for reliable identification of both oxacillin-susceptible and oxacillin-resistant Staphylococcus aureus.

A new latex agglutination test (Pastorex Staph-Plus, Sanofi Diagnostics Pasteur), consisting of a mixture of latex particles coated with fibrinogen and immunoglobulin G for the detection of clumping factor and protein A and latex particles sensitized with monoclonal antibodies directed to Staphylococcus aureus serotype 5 and 8 capsular polysaccharides, was compared with three commercially available rapid agglutination methods for the identification of 220 isolates of S. aureus (61 oxacillin resistant) and 128 isolates of coagulase-negative staphylococci. The sensitivity for identification of S. aureus was high with the Pastorex Staph-Plus test (98.6%) compared with those of the other tests, which ranged from 91.8 to 84.5%. Test sensitivities for the identification of oxacillin-resistant S. aureus were as follows: Pastorex Staph-Plus, 95.1%; Pastorex Staph, 73.8%; Staphyslide, 72.1%; and StaphAurex, 49.2%.

Antibodies, Monoclonal↗