Comparison of agar dilution, broth microdilution, E-test, disk diffusion, and automated Vitek methods for testing susceptibilities of Enterococcus spp. to vancomycin

An evaluation was undertaken to determine the optimal method for testing the susceptibilities of 100 clinical isolates and two reference strains of Enterococcus spp. to vancomycin in vitro. Six testing methods were studied by using the following media and incubation times: agar screen with the Synergy Quad Plate (Remel, Lenexa, Kans.), an in-house-prepared brain heart infusion (BHI) agar plate, and an in-house-prepared Mueller-Hinton (MH) agar plate, all incubated for 24 or 48 h; broth microdilution (Sensititre Just One Strip; AccuMed International, Inc., West Lake, Ohio) with BHI or cation-adjusted MH broth incubated for 24 or 48 h; agar dilution with BHI or MH agar incubated for 24 or 48 h; epsilometer test (E test; AB BioDisk, Solna, Sweden) with BHI or MH agar incubated for 24 or 48 h; disk diffusion with BHI or MH agar incubated for 24 or 48 h; and the automated Vitek method with the gram-positive susceptibility Staphylococcus aureus card and R02.03 software (bioMerieux, Inc., Hazelwood, Mo.). Growth failures occurred with MH media (n = 6) but not with BHI media. One growth failure occurred with the Vitek method. Results for each testing method for each Enterococcus strain were interpreted as susceptible, intermediate, or resistant according to current National Committee for Clinical Laboratory Standards (NCCLS) criteria and compared to the vancomycin resistance genotype (i.e., vanA, vanB, vanC-1, or vanC-2/3). For all methods, extension of the incubation time from 24 h to 48 h either produced no difference in the results or gave poorer results. The following methods produced no very major or major interpretive errors: broth microdilution with BHI media incubated for 24 h, agar dilution with BHI media incubated for 24 or 48 h, and E test with BHI media incubated for 24 or 48 h. Unacceptable frequencies of very major errors (> 1%) occurred with all methods for which MH media were used. Minor interpretive errors were frequent with all methods. These minor interpretive errors also occurred most frequently with Enterococcus strains with vanC genes, which encoded low-level vancomycin resistance (MIC < or = 8 microg/ml), as opposed to Enterococcus strains which possessed vanA or vanB genes, which encoded higher-level vancomycin resistance (MIC > or = 64 microg/ml). Modification of NCCLS breakpoints, especially for motile Enterococcus spp. (E. casseliflavus, E. flavescens, and E. gallinarum), may resolve this problem; however, in the current study, one E. faecalis strain and one E. faecium strain carried only the vanC gene. The agar screen method may also require reformulation. The current agar screen plate contains 6 microg of vancomycin per ml, which may not detect all low-level resistance associated with vanC genotypes. Nevertheless, the clinical significance of this low-level vancomycin resistance remains unknown.     

Comparison of agar dilution, broth dilution, disk diffusion, and the E-test for susceptibility testing of penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae

An evaluation to determine the optimal methods for the in vitro susceptibility testing of 41 clinical isolates and the ATCC 49619 strain of Streptococcus pneumoniae to penicillin was undertaken. No very major or major interpretive errors were observed with the following test methods and media: agar dilution using either Mueller-Hinton medium with lysed horse blood or Haemophilus test medium; broth dilution using cation-adjusted Mueller-Hinton medium with lysed horse blood, Haemophilus test medium, or Todd-Hewitt medium; and the epsilo-meter test (E-test) using agar containing Mueller-Hinton medium and 5% sheep blood. The disk diffusion method using agar containing Mueller-Hinton medium and 5% sheep blood agar was an effective screening method, requiring confirmation by a dilution susceptibility test method.     

Antipneumococcal activities of RP 59500 (quinupristin-dalfopristin), penicillin G, erythromycin, and sparfloxacin determined by MIC and rapid time-kill methodologies

Previous time-kill studies have shown that RP 59500 is rapidly bactericidal against pneumococci. To extend these findings, the activities of RP 59500, its two components RP 57669 RP 54476, penicillin G, erythromycin and sparfloxacin against 26 penicillin-susceptible, 25 penicillin-intermediate, and 25 penicillin-intermediate, and 25 penicillin-resistant pneumococci were determined by the agar dilution MIC and the time-kill testing methodologies within 10 min (ca. 0.2 h) and at 1 and 2 h. Respective agar dilution MICs at which 90% of isolates are inhibited for penicillin-susceptible, -intermediate, and -resistant strains were as follows: penicillin G, 0.03, 1, and 4 micrograms/ml;RP 59500, 1, 1, and 1 microgram/ml; RP 57669, 8, 32, and 16 micrograms/ml; RP 54476, > 128, > 128, and > 128 micrograms/ml; erythromycin, 0.06, 2, and > 128 micrograms/ml; and sparfloxacin, 1, 0.5, and 0.5 microgram/ml. RP 59500 was equally active (MIC at which 90% of isolates are inhibited, 1.0 microgram/ml) against erythromycin-susceptible and -resistant strains. Time-kill testing results showed that only RP 59500 at one to four times the MIC killed pneumococci at 0.2 h; RP 59500 was also the most active compound at 1 and 2 h. By comparison, penicillin and sparfloxacin at one, two, and four times the MICs reduced the original inoculum by > or = 1 log at 2 h for 46, 80, and 95% and for 50, 72, and 86% of strains, respectively. The killing activity of RP 59500 was the same against erythromycin-susceptible and -resistant strains. RP 57669, RP 54479, and erythromycin were either inactive or bacteriostatic at 2 h. Of all drugs tested, RP 59500 yielded the most rapid killing.     

Antibiotic susceptibility testing (agar disk diffusion and agar dilution) of clinical isolates of Corynebacterium jeikeium

Thirty-three clinical isolates of Corynebacterium jeikeium were examined for susceptibility to 27 antimicrobial drugs with the agar dilution test. Sheep-blood-supplemented Mueller-Hinton agar performed better than Wilkins-Chalgren agar. Disk susceptibility (Bauer-Kirby) tests were carried out in parallel with 24 of the chemotherapeutic agents. All isolates were susceptible to teicoplanin and vancomycin. All isolates resisted fosfomycin, mupirocin, and trimethoprim-sulfamethoxazole. The isolates varied in susceptibility to ciprofloxacin, doxycycline, fusidic acid, ofloxacin, and tetracycline; most were susceptible to rifampin. Surprisingly few discrepancies between agar dilution and disk diffusion tests were encountered when utilizing NCCLS interpretive criteria currently valid for enterococcal isolates.     

Comparison of agar dilution, disk diffusion, MicroScan, and Vitek antimicrobial susceptibility testing methods to broth microdilution for detection of fluoroquinolone-resistant isolates of the family Enterobacteriaceae

Fluoroquinolone resistance appears to be increasing in many species of bacteria, particularly in those causing nosocomial infections. However, the accuracy of some antimicrobial susceptibility testing methods for detecting fluoroquinolone resistance remains uncertain. Therefore, we compared the accuracy of the results of agar dilution, disk diffusion, MicroScan Walk Away Neg Combo 15 conventional panels, and Vitek GNS-F7 cards to the accuracy of the results of the broth microdilution reference method for detection of ciprofloxacin and ofloxacin resistance in 195 clinical isolates of the family Enterobacteriaceae collected from six U.S. hospitals for a national surveillance project (Project ICARE [Intensive Care Antimicrobial Resistance Epidemiology]). For ciprofloxacin, very major error rates were 0% (disk diffusion and MicroScan), 0.9% (agar dilution), and 2.7% (Vitek), while major error rates ranged from 0% (agar dilution) to 3.7% (MicroScan and Vitek). Minor error rates ranged from 12.3% (agar dilution) to 20.5% (MicroScan). For ofloxacin, no very major errors were observed, and major errors were noted only with MicroScan (3.7% major error rate). Minor error rates ranged from 8.2% (agar dilution) to 18.5% (Vitek). Minor errors for all methods were substantially reduced when results with MICs within +/-1 dilution of the broth microdilution reference MIC were excluded from analysis. However, the high number of minor errors by all test systems remains a concern.     

Antibiotic susceptibility testing (agar disk diffusion and agar dilution) of clinical isolates of Enterococcus faecalis and E. faecium: comparison of Mueller-Hinton, Iso-Sensitest, and Wilkins-Chalgren agar media

Forty-two isolates of Enterococcus faecalis and 56 isolates of Enterococcus faecium, including 8 vancomycin-resistant strains, were examined for comparative susceptibility to 27 antimicrobial drugs with the agar dilution method, employing Mueller-Hinton (MHA), Iso-Sensitest (ISTA), and Wilkins-Chalgren (WCA) agar. The Bauer-Kirby agar disk diffusion method was used to comparatively test 24 of the agents in parallel. The enterococci yielded better growth on ISTA and WCA. However, WCA completely antagonized co-trimoxazole and, though less, fosfomycin. Importantly, WCA slightly reduced the activities of teicoplanin (minimal inhibitory concentrations, MICs, raised up to twofold) and vancomycin (MICs raised two- to fourfold) against enterococci and staphylococcal quality control strains. Therefore, WCA was judged unsuitable for susceptibility testing of enterococci. For E. faecalis no discrepancies between agar dilution MICs and inhibition zone diameters were encountered with augmentin, ampicillin, ampicillin-sulbactam, chloramphenicol, mupirocin, oxacillin, teicoplanin, and co-trimoxazole. Overall, MHA yielded fewer very major (category I) and major (category II) discrepancies than ISTA. However, numerous minor (category III), slight (category IV), minimal (category V), and/or negligible (category VI) discrepancies were encountered with ciprofloxacin, doxycycline, erythromycin, fosfomycin, fusidic acid, meropenem, ofloxacin and rifampin. With respect to E. faecium, only cefotaxime, mupirocin, oxacillin, and teicoplanin yielded nondiscrepant results. Several very major (I) and major (II) discrepancies were observed with augmentin, ampicillin, ampicillin-sulbactam, doxycycline, fusidic acid, imipenem, and penicillin G. Minor discrepancies (categories III-VI) were particularly numerous with augmentin, chloramphenicol, ciprofloxacin, doxycycline, and piperacillin. The largest numbers of negligible (VI) discrepancies were noted with fosfomycin, fusidic acid, and ofloxacin. It is recommended to test one cephalosporin (cefuroxime or the like) in parallel for educational purposes and to exclude fosfomycin, fusidic acid, and rifampin from test batteries because of the wide scatter of test results. The large number of minimal (V) discrepancies of ciprofloxacin against E. faecalis, the numerous minor (III) and slight (IV) discrepancies of chloramphenicol against E. faecium, and the not insignificant number of very major (I) and minor (III) discrepancies observed with meropenem against isolates of E. faecalis necessitated proposals for new disk intermediate susceptibility criteria.     

Comparison of antimicrobial susceptibilities of Corynebacterium species by broth microdilution and disk diffusion methods

Corynebacterium species are increasingly being implicated in foreign-body infections and in immunocompromised-host infections. However, there are no specific recommendations on the method or the criteria to use in order to determine the in vitro activities of the antibiotics commonly used to treat Corynebacterium infections. The first aim of our study was to compare the susceptibilities of various species of Corynebacterium to vancomycin, erythromycin, and penicillin by using a broth microdilution method and a disk diffusion method. Second, the activity of penicillin against our isolates was assessed by using the interpretative criteria recommended by the National Committee for Clinical Laboratory Standards for the determination of the susceptibility of streptococci and Listeria monocytogenes to penicillin. Overall, 100% of the isolates were susceptible to vancomycin, while considerable variations in the activities of erythromycin and penicillin were noted for the different species tested, including the non-Corynebacterium jeikeium species. A good correlation in the susceptibilities of vancomycin and erythromycin between the disk diffusion and the microdilution methods was observed. However, a 5% rate of major or very major errors was detected with the Listeria criteria, while a high rate of minor errors (18%) was noted when the streptococcus criteria were used. Our findings indicate considerable variations in the activities of erythromycin and penicillin against the various species of Corynebacterium. Because of the absence of definite recommendations, important discrepancies were observed between the methods and the interpretations of the penicillin activity.     

Evaluation of the standardized disk diffusion and agar dilution antibiotic susceptibility test methods by using strains of Neisseria gonorrhoeae from Tucumán, Argentina

At present, most Neisseria gonorrhoeae testing is done with beta-lactamase and agar dilution tests using common therapeutic agents. Generally, in bacteriological diagnosis laboratories in Argentina, study of antibiotic susceptibility of N. gonorrhoeae is based on beta-lactamase determination and agar dilution method using common therapeutic agents. The National Committee for Clinical Laboratory Standards (NCCLS) recently described a disk diffusion test that produces results similar to the reference agar dilution method for antibiotic susceptibility of N. gonorrhoeae. We obtained 57 gonococcal isolates from patients attending a clinic for sexually transmitted diseases in Tucumán, Argentina. Antibiotic susceptibility tests using agar dilution and disk diffusion techniques were compared. The established NCCLS interpretive criteria for both susceptibility methods appeared to be applicable to domestic gonococcal strains. The correlation between the minimum inhibitory concentration (MIC's) and the zones of inhibition was studied for penicillin, ampicillin, cefoxitin, spectinomycin, cefotaxime, cephaloridine, cephalexin, tetracycline, norfloxacin and kanamycin. Dispersion diagrams showed a high correlation between both methods, with a sensitivity of 89% and specificity of 91%.     

Evaluation of mannitol salt agar for detection of oxacillin resistance in Staphylococcus aureus by disk diffusion and agar screening

Mannitol salt agar was evaluated for detection of oxacillin resistance in 136 Staphylococcus aureus isolates. All mecA-positive isolates (n = 54) were correctly categorized as oxacillin resistant by the disk diffusion test (1-microgram disk; zone diameter, < 16 mm); the specificity was 97.6%. Agar screening (2 micrograms of oxacillin per ml) revealed a sensitivity of 98.1% and a specificity of 95.1%.     

Assessment of the synergistic interactions of levofloxacin and ampicillin against Enterococcus faecium by the checkerboard agar dilution and time-kill methods

Multidrug-resistant enterococci have become increasingly difficult to eradicate in a growing number of nosocomial infections. With the emergence of vancomycin-resistant enterococci, the use of synergistic antibiotic combinations has become one of the only remaining therapeutic options. Levofloxacin, the active l-isomer of ofloxacin, is a new oral and intravenous fluoroquinolone with a broad spectrum of activity against numerous Gram-positive, Gram-negative, and atypical organisms. The in vitro activity of levofloxacin, alone and in combination with ampicillin, against recent clinical isolates of Enterococcus faecium was assessed for synergistic interactions using the checkerboard agar dilution technique and time-kill methodology. Against all strains, the static technique of checkerboard agar dilution demonstrated indifferent or additive effects for the ampicillin + levofloxacin combination. With the dynamic time-kill technique, synergy was demonstrated for ampicillin (16 micrograms/ml) + levofloxacin (2 micrograms/ml) combination against three levofloxacin-sensitive, ampicillin-resistant isolates. At 24 h, the combination yielded a > or = 2-log10 decrease in CFU/ml compared to levofloxacin alone, while ampicillin had negligible effects. Against both a levofloxacin-intermediate, ampicillin-resistant isolate, and a highly levofloxacin-resistant, ampicillin-resistant isolate, none of the ampicillin+levofloxacin combinations tested demonstrated a synergistic interaction. The time-kill method suggested synergy for the ampicillin+levofloxacin combination against some strains of E. faecium.     

Comparison of broth microdilution method using Haemophilus test medium and agar dilution method for susceptibility testing of Eikenella corrodens

Susceptibility testing of Eikenella corrodens is usually performed by a Mueller-Hinton sheep blood agar dilution (AD) method. However, this method is impractical for testing only a few strains. We compared AD with the broth microdilution method using Haemophilus test medium (HTM) in order to determine the susceptibility of 36 clinical isolates of E. corrodens to eight antimicrobial agents. MICs obtained by the HTM method yielded 95.5 and 84% agreement (within 2 and 1 log2 dilutions, respectively) with those obtained by AD. The HTM method with incubation in CO2 for 48 h was highly reproducible and constitutes an easy alternative for antimicrobial susceptibility testing of E. corrodens.     

Comparison of broth macrodilution, broth microdilution and E-test susceptibility tests of Cryptococcus neoformans for fluconazole

Forty Cryptococcus neoformans strains isolated from cerebral spinal fluid specimens collected from 39 patients were included in the study. The MICs for fluconacole were determined by YNB macrodilution test, microdilution tests using both RPMI1640 and YNB medium and E-tests on solidified RPMI1640 medium, Casitone and YNB agar. In comparison with the reference macrodilution method NCCLS M27-P both the microdilution as well as the E-test techniques can be used for fluconacole susceptibility testing of Cr. neoformans.     

Paramyxovirus phosphoproteins form homotrimers as determined by an epitope dilution assay, via predicted coiled coils

When HA epitope-tagged and untagged Sendai virus (SeV) P proteins are coexpressed and the products reacted with anti-HA, the untagged P protein is also selected because this protein is found as an oligomer. The oligomer was determined to be a homotrimer by coselection studies in which increasing amounts of untagged versus tagged protein were coexpressed, and these findings were extended to mumps virus, a member of the rubulavirus genus. The region of the SeV protein responsible for the oligomerization was localized to residues 344-411. Computer analysis of the 13 Paramyxovirus P proteins in the database revealed that all but one are predicted to form coiled coils in this region, the first of only two regions that can be aligned throughout the entire virus subfamily. The predicted coiled-coil region of the measles virus P protein, when grafted onto the C-terminus of the normally monomeric La protein, led to the efficient oligomerization of this reporter protein. The predicted coiled-coil region of these P proteins thus appears to be sufficient for oligomerization.     

Studies on bactericidal activities of beta-lactam antibiotics on agar plates: the correlation with the antibacterial activities determined by the conventional methods

A simplified method to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of beta-lactam antibiotics on agar plates is described. MIC values were determined on agar plates for benzylpenicillin, methicillin and cephalothin using Staphylococcus aureus and Klebsiella pneumoniae. A beta-lactamase solution was then sprayed onto the plates to inactivate the drug(s). After further incubation at 37 degrees C overnight, the minimal concentration at which no test bacteria were visible on the plates was defined as MBC. Both MIC and MBC values decreased with decreased inoculum size. The two values were almost coincidental when high dilutions were used as the inocula. These values were compared with those obtained by the conventional broth dilution method. In this study, MIC as well as MBC values determined by the simplified method were generally smaller than the values determined by the broth dilution technique.     

Quality control limits for dilution and disk diffusion susceptibility tests of trovafloxacin against eight quality control strains

A 10-laboratory collaborative effort was designed to generate data to propose quality control limits for susceptibility tests of trovafloxacin. Broth microdilution, agar dilution, and disk diffusion tests were evaluated with eight different control strains. All tests were reproducible, and control limits are proposed.     

A multicenter, investigator-blinded, randomized comparison of oral levofloxacin and oral clarithromycin in the treatment of acute bacterial sinusitis

The effects of Vitamin E administration on antioxidant enzyme activities and nitrite-nitrate levels of the reperfused rat kidney tissues were investigated by performing a 60 min ischemia followed by 24 and 72 hours of reperfusion. Vitamin E administration or the placebo (SF) was applied as 100 mg/kg BW. As expected, catalase (CAT) (p<0.05) and superoxide dismutase (SOD) (p<0.05) activities of ischemia/reperfused (I/R) kidney tissue were lower and malondialdehyde (MDA) levels were higher than control kidneys in both SF and vitamin E treated groups following 24 h reperfusion. During reperfusion of long term (72 h), vitamin E triggered a decrease in the MDA levels in the ischemic tissue, while it did not provoke a significant effect on SOD and catalase activities. Total nitrite levels of ischemic tissues in both of the groups were higher than matched control kidneys and this elevation was more clear in the vitamin E treated group. Our results showed that vitamin E has a protective effect on I/R injury, by a direct chain breaking effect on lipid peroxidation (LPO) and hence preventing the nitric oxide (NO) reservoir of ischemic tissue. Alfa-tocopherol may be a promising agent for the prevention of tissue injury caused by free oxygen radicals.     

Volume of extravascular lung fluid determined by blood ultrasound velocity and electrical impedance dilution

A hypertonic sodium chloride bolus passing through the lung has a sound velocity transient that is biphasic when it reaches the carotid artery. This transient is compatible with water moving into the hypertonic bolus from the lung parenchyma, thereby leaving the lung parenchyma hypertonic. Subsequently, as the bolus leaves the lung vasculature, water passes from the blood into the tissue to return the lung tonicity to baseline, giving a moment when net movement is zero, an instant of osmotic equilibrium. Concurrent measurements of impedance track the sodium chloride transient. A theoretic basis for the calculation of extravascular lung water is derived from the water transferred to the blood, the amount of sodium chloride moved from blood to the lung, and the increase in blood osmolarity measured at the moment of equilibrium. Examples from measurements on sheep suggest that two intravenous injections of hypertonic and isotonic sodium chloride, with observations of sound velocity and electrical impedance in the systemic arterial circulation (which could also provide the cardiac output), provide a basis for calculation of lung permeability, water and salt movements, and extravascular lung water estimation.     

Postantibiotic effect and postantibiotic sub-MIC effect of levofloxacin compared to those of ofloxacin, ciprofloxacin, erythromycin, azithromycin, and clarithromycin against 20 pneumococci

The postantibiotic effect (PAE) (10 times the MIC of quinolones, 5 times the MIC of macrolides) and postantibiotic sub-MIC effect (PAE-SME) at 0.125, 0.25, and 0.5 times the MIC were determined for levofloxacin, ciprofloxacin, ofloxacin, erythromycin, azithromycin, and clarithromycin against 20 pneumococci. Quinolone PAEs ranged between 0.5 and 6.5 h, and macrolide PAEs ranged between 1 and 6 h. Measurable PAE-SMEs (in hours) at the three concentrations were 1 to 5, 1 to 8, and 1 to 8, respectively, for quinolones and 1 to 8, 1 to 8, and 1 to 6, respectively, for macrolides.     

In vitro activities of azithromycin, clarithromycin, erythromycin, and tetracycline against 13 strains of Chlamydia pneumoniae

Thirteen strains of Chlamydia pneumoniae were evaluated for their in vitro susceptibilities to azithromycin, clarithromycin, erythromycin, and tetracycline. The MIC ranges were 0.125 to 0.5 micrograms/ml for azithromycin, 0.031 to 1.0 micrograms/ml for clarithromycin, 0.125 to 1.0 micrograms/ml for erythromycin, and 0.25 to 1.0 micrograms/ml for tetracycline. The ranges for the minimal lethal concentrations were 0.125 to 0.5 micrograms/ml for azithromycin, 0.031 to 1.0 micrograms/ml for clarithromycin, 0.125 to 1.0 micrograms/ml for erythromycin, and 0.25 to 1.0 micrograms/ml for tetracycline. Clarithromycin and azithromycin were the most active antibiotics against C. pneumoniae in vitro.