Interpretive criteria of ceftibuten disk diffusion susceptibility tests according to the DIN 58 940 method

Objective   This study aimed to establish interpretive criteria for agar diffusion tests with ceftibuten disks according to DIN standards.
Methods   Minimal inhibitory concentrations (MICs) and inhibition zones produced by ceftibuten in the disk diffusion test were determined for 275 recent bacterial isolates, including 11 species with 25 strains each. Regression analysis was performed for two disk loads (10 µg and 30 µg).
Results   Correlation of MICs and zone diameters was good, with correlation coefficients of r  = − 0.97 for both tested disk loads. Evaluation of the calculated zone size criteria for all species showed no very major discrepancies or no major discrepancies. The 30-µg disks, however, produced unacceptably large inhibition zones for very susceptible strains, so that usage of 10-µg disks must be recommended when testing according to DIN standards.
Conclusion   Based on the MIC breakpoints recommended by the DIN (≥8 mg/L and ≤ 1 mg/L), the following interpretive breakpoints for disk diffusion susceptibility tests with 10-µg ceftibuten disks were calculated using regression line analysis: ≤19 mm for resistance and ≥ 27 mm for susceptiblity. Proposed inhibition zone diameters for the reference strain Escherichia coli ATCC 25922 are between 31 and 36 mm.     

Tentative interpretive criteria for in vitro antibacterial susceptibility testing with imipenem

Imipenem is a member of a new class of highly potent beta-lactam antibiotics, carbapenems, with a very broad antibacterial spectrum. This study was undertaken to determine tentative interpretive criteria for in vitro susceptibility testing with 10-micrograms imipenem disks. A careful examination of the zone diameters and the corresponding MICs for 489 clinical isolates by regression-line analysis and the error rate-bounded classification scheme suggested the following guidelines: greater than or equal to 16 mm with an MIC correlate of less than or equal to 4 micrograms/ml for susceptible, 14 to 15 mm (8 micrograms/ml) for moderately susceptible, and less than or equal to 13 mm (greater than or equal to 16 micrograms/ml) for resistant. Lack of cross-resistance between imipenem and broad-spectrum cephalosporins such as cefotaxime and ceftazidime argues against their use as class disks to predict in vitro susceptibility of bacterial species to carbapenems.     

Discrepancies in Weil-Felix and microimmunofluorescence test results for Rocky Mountain spotted fever.

Only 4.2% of 284 single specimens and 17.6% of 51 pairs of sera reactive in Weil-Felix agglutination tests for Rocky Mountain spotted fever were confirmed by a specific Rickettsia rickettsii microimmunofluorescence test.     

Evaluation of three broth disk methods for testing the susceptibility of anaerobic bacteria to imipenem

Imipenem is a member of a new class of highly potent beta-lactam antibiotics, carbapenems, with an antibacterial spectrum that includes nearly all currently known aerobic and anaerobic bacterial species of clinical significance. Although relatively stable in most standard laboratory media used for antimicrobial susceptibility testing, imipenem undergoes rapid inactivation in thioglycolate broth, a recommended medium for susceptibility testing of anaerobic bacteria by the broth disk method. In the current study, a panel of 36 anaerobic bacteria consisting of 28 clinical isolates and eight quality control strains was used to determine the suitability and accuracy of the broth disk methods with brain heart infusion, Schaedler, and anaerobic broths, in comparison to the reference agar dilution method, for the anaerobic susceptibility testing of imipenem. To achieve single test concentrations of approximately 8, 16, and 64 micrograms/ml for imipenem, cefoxitin, and piperacillin, respectively, which correspond to the MIC breakpoints of the test drugs, four 10-microgram imipenem disks, three 30-microgram cefoxitin disks, and three 100-microgram piperacillin disks were used in 5 ml of broth. The correlation between the reference agar dilution method and each of the three broth disk elution procedures evaluated was excellent, for imipenem (100% agreement) and somewhat less so for cefoxitin and piperacillin. Therefore, brain heart infusion, Schaedler, and anaerobic broths, but not thioglycolate broth, are suitable for anaerobic susceptibility testing of imipenem by the disk elution method.     

A system for reporting quantitative antimicrobic susceptibility test results.

A reporting system for the laboratory to communicate quantitative antimicrobic susceptibility test results to the physician in a manner that will optimize proper utilization of such test results is described. Minimal inhibitory concentrations are reported on a scale of clinically achievable concentrations, thus indicating the degree of susceptibility.     

Discrepancies between results by E-test and standard microbroth dilution testing of Streptococcus pneumoniae for susceptibility to vancomycin.

Vancomycin susceptibility testing of Streptococcus pneumoniae by the E-test consistently resulted in MICs that were at the upper limit of the 1-microgram/ml susceptible category defined by current National Committee for Clinical Laboratory Standards guidelines and were always higher than MICs obtained by microbroth dilution. Three of five E-test results for S. pneumoniae ATCC 49619 were higher than the acceptable limits.     

Quality control for antimicrobial susceptibility test using correlation between MIC results

The method of National Committee for Clinical Laboratory Standards (NCCLS) is widely used for the daily quality control of the antimicrobial susceptibility test. This method, however, cannot detect the accidental error, although it is useful to detect the systematic error in the examination. We developed a computer program using the correlation between the various antimicrobial susceptibility test results to detect an accidental error. The combinations of the MIC results determined for two antimicrobial agents which showed a high correlation coefficient (> or = 0.7), were selected from 98 bacterial species (2122 strains) isolated from January 2000 to December 2000 at Oita Medical University Hospital. Subsequently, a total of 127 combinations of antimicrobial agents for 13 species were selected on the basis of acceptable correlation ranges. Then, the method were verified with 666 strains (5753 combinations) isolated during the period of January to June, 2001. Twenty-six strains (47 combinations) were identified as an unexpected result, and the occurrence of error were confirmed in 3 strains (12 combinations). These results suggest that this method which evaluated the correlation between MICs against different antimicrobial agents is applicable for the quality control of antimicrobial susceptibility testings.     

Comparison of susceptibility test methods to detect penicillin susceptibility in Streptococcus pneumoniae isolates

The increasing prevalence of penicillin-resistant Streptococuus pneumoniae urges for fast and accurate susceptibility testing methods. This study evaluated the comparability of three commonly used techniques; disk diffusion, E-test and agar dilution, to detect penicillin susceptibility in clinical isolates of S. pneumoniae. Fifty pneumococcal isolates, obtained from patients at the University of Malaya Medical Centre, were selected to include both penicillin-susceptible strains and those that had decreased susceptibility (resistant and intermediate) to penicillin. The minimum inhibitory concentration (MIC) values of penicillin to serve as the reference was determined by the agar dilution method in which, based on the MIC breakpoints recommended by the National Committee for Clinical Laboratory Standards (NCCLS), 27 strains had decreased susceptibility to penicillin with 17 strains resistant and 10 intermediate. Comparing to the agar dilution method, oxacillin disk diffusion test detected all strains with decreased penicillin susceptibility as such while E-test showed a close agreement of susceptibility (92%) of the isolates to penicillin. This confirmed that oxacillin is a good screening test for S. pneumoniae isolates with decreased susceptibility to penicillin while E-test is very reliable for rapid and accurate detection of penicillin susceptibility.     

Multicenter evaluation of use of penicillin and ampicillin as surrogates for in vitro testing of susceptibility of enterococci to imipenem

Imipenem is approved by the U.S. Food and Drug Administration (FDA) for treatment of infections caused by Enterococcus faecalis. However, there are no NCCLS guidelines for testing susceptibility of enterococci against imipenem. To assess whether or not ampicillin or penicillin could be used as a surrogate for broth microdilution (BMD) testing of imipenem versus Enterococcus species, 633 strains of E. faecalis, E. faecium, and other enterococci isolated from blood cultures of patients at three geographically distinct university hospitals were tested by the NCCLS BMD and disk diffusion (DD) methods. Using FDA susceptibility breakpoints for imipenem and NCCLS breakpoints for penicillin and ampicillin, categorical agreement (CA) for penicillin-imipenem and ampicillin-imipenem tested with E. faecalis and E. faecium by BMD was >/=94% but was /=98% and was 92% for other enterococci; CA for penicillin-imipenem was 91% for E. faecalis, 98% for E. faecium, and 87% for other enterococci. Further analysis showed that testing E. faecalis with ampicillin resulted in no false-susceptible (FS) or false-resistant (FR) results by BMD, no FS results by DD, and a single FR result by DD (0.2%), whereas testing with penicillin resulted in no FS results by BMD or DD and two FR results by BMD (0.4%). For E. faecium and other enterococci, the combination of FS and FR results was such that surrogate testing with penicillin or ampicillin appears not to be sufficiently reliable to be used clinically. We conclude that ampicillin is an accurate predictor of the in vitro activity of imipenem against E. faecalis.     

Comparison of cefadroxil and cephalothin disk susceptibility test results.

Diffusion susceptibility tests with 30-micrograms cefadroxil disks and 30-micrograms cephalothin disks were evaluated. For both agents, the same zone size interpretive criteria were recommended (less than or equal to 14 mm for resistance and greater than or equal to 18 mm for susceptibility). Tests were performed with 904 bacterial isolates, and the data were examined to determine whether the two cephalosporins might be used interchangeably for purposes of in vitro susceptibility testing. When Haemophilus influenzae, Listeria monocytogenes, and methicillin-resistant staphylococci were evaluated, the two agents differed significantly. For testing other species, a cephalothin disk or cephalothin MIC could be used for predicting susceptibility or resistance to cefadroxil.     

Tiamulin activity against fastidious and nonfastidious veterinary and human bacterial isolates: initial development of in vitro susceptibility test methods

Tiamulin is a pleuromutilin derivative used in veterinary practice for the control and specific therapy of infections in swine. This report summarizes studies to establish standardized susceptibility testing methods, interpretive criteria, and reagent details for use in veterinary methods recently developed by the National Committee for Clinical Laboratory Standards (NCCLS) (standards M31-A and M37-A, NCCLS, Wayne, Pa., 1999). A total of 636 fastidious and nonfastidious animal and human pathogens were processed by using media and procedures described by the NCCLS. Tiamulin disk diffusion tests used a 30-microg disk concentration, and the proposed MIC breakpoints corresponding to levels achievable in animal target tissues (lung) were < or =4 microg/ml for susceptibility and > or =32 microg/ml for resistance. Correlate zone diameters for specific nonfastidious species were as follows: for Pasteurella multocida and staphylococci tested on Mueller-Hinton agar, susceptibility at > or =19 mm and resistance at < or =11 mm, and for Actinobacillus suis, Erysipelothrix rhusiopathiae, and Streptococcus suis tested on enriched chocolate Mueller-Hinton agar, susceptibility at > or =16 mm and resistance at < or =8 mm. When Actinobacillus pleuropneumoniae was tested, a susceptibility breakpoint of < or =16 microg/ml (> or =9 mm) was suggested for veterinary fastidious medium broth and enriched chocolate Mueller-Hinton agar. Absolute categorical agreement between NCCLS dilution and disk diffusion test results with these criteria ranged from 90.5 to 96.2%. Tiamulin susceptibility testing methods appear to be accurate in their categorical classification for indicated species, and their availability will allow immediate testing of animal isolates to guide therapy via appropriate levels of dosing and to monitor the development of resistance for agents in this unique class.     

Assessment of genetic damage by methyl methacrylate employing in vitro mammalian test system

Methyl methacrylate (MMA) is a volatile liquid widely used in the manufacture of acrylic polymers. In modern dentistry, MMA is the mainstream material in denture bases. MMA has been implicated as primary irritant and sensitizer, which can cause allergic eczematous reaction on the oral mucosa as well as skin. To date, there is growing concern that MMA may produce genetic damage by inducing mutation. In this study, colony forming efficiency, DNA synthesis, and cytogenetic assays were performed to investigate the adverse effects of MMA in cultured CHO cells. MMA was found to decrease colony formation in a dose- and time-dependent manner (P<0.05). MMA also inhibited DNA synthesis in a dose-dependent manner (P<0.05). The chromosome aberrations induced by MMA were the chromatid-type aberrations in the treated cultures. Moreover, the gaps and breaks were the most common type of aberrations observed. The sister-chromatid exchange frequencies were found to increase in the concentration of MMA. In this study, MMA was found to be not only a cytotoxic agent but also a genotoxic agent. The effects observed following treatment with low dose for longer duration is of relevance to the condition of the oral mucosa of the denture wears. Denture base resin could constantly release MMA extended periods, possibly causing moderate toxic reactions and possibly contributing to adverse effects on the mucosa.     

Assessment of two commercial susceptibility test methods for determination of daptomycin MICs

Daptomycin is a lipopeptide antibiotic with activity against several important gram-positive bacterial pathogens, including drug-resistant staphylococci and enterococci. Because the mechanism of action of daptomycin is calcium-dependent depolarization of the cell membrane, susceptibility testing requires medium supplemented with a physiological level of calcium. This study assessed two Food and Drug Administration-cleared commercial test devices for determination of daptomycin MICs, Etest and JustOne. A collection of 220 selected isolates, including Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, E. faecium, E. avium, E. durans, E. casseliflavus, and E. gallinarum, were tested by both methods. Included in the collection were 22 S. aureus and 14 Enterococcus sp. isolates that were recovered from patients and were nonsusceptible on the basis of the daptomycin MICs. As the reference method for comparison, all isolates were tested by the Clinical and Laboratory Standards Institute broth microdilution method incorporating cation-adjusted Mueller-Hinton broth with 50 microg/ml calcium. Daptomycin MICs agreed, within 1 twofold dilution, for 97% of the isolates by Etest and for 100% by JustOne. However, daptomycin MICs determined by Etest were 1 dilution lower than the reference MICs for 65% of the Enterococcus sp. isolates tested. This resulted in 28.5% very major (VM) errors (4/14) with enterococci (all E. faecium) but none (0/22) with staphylococci. Use of JustOne yielded MICs that were 1 dilution lower than the reference MICs for 69% of the staphylococci and 25% of the enterococci. This resulted in 13.6% VM errors (3/22) with staphylococci and 14.3% VM errors (2/14) with enterococci. The manufacturer-recommended JustOne inoculum preparation resulted in mean colony counts of only 5 x 10(4) to 1 x 10(5) CFU/ml in the wells of the strip. Increasing the inoculum to 3 x 10(5) to 4 x 10(5) CFU/ml eliminated two of five VM errors upon retesting. No major interpretive errors occurred with either device. In summary, daptomycin MICs generated by the Etest or JustOne method generally agreed within 1 dilution of the reference daptomycin MICs. However, both devices produced slightly lower MICs that resulted in some VM errors.     

Correlation of in vitro susceptibility results for amoxicillin-clavulanate and ampicillin-sulbactam tested against Escherichia coli.

The results of amoxicillin-clavulanate (AUG) and ampicillin-sulbactam (A/S) susceptibility testing by three different susceptibility testing methods, the MicroScan, Etest, and Kirby-Bauer methods, for 61 consecutive isolates of ampicillin-resistant Escherichia coli from different patients were compared. There was poor correlation of results for the two agents, the most and least marked discrepancies being observed by the MicroScan method (86.9% susceptible to AUG and 4.9% susceptible to A/S) and the Kirby-Bauer method (39.4% susceptible to AUG and 32.8% susceptible to A/S), respectively. More organisms were susceptible to AUG than A/S, regardless of the susceptibility testing methodology. The results from a College of American Pathologists survey with one E. coli isolate tested at different institutions also indicated greater susceptibility to AUG than to A/S. These agents are thought to be equally efficacious clinically. The discrepancies observed among methods for each antimicrobial inhibitor combination and the discrepancies observed between the two agents by each testing method suggest that the breakpoints for these agents need to be reevaluated.     

Discrepancy in antimicrobial susceptibility test results obtained for oral streptococci with the Etest and agar dilution

A total of 270 viridans group streptococci (VS) isolated from healthy children, identified to the species level, were tested for their susceptibilities to penicillin, imipenem, erythromycin, and vancomycin. A total of 270 isolates and 1,080 organism-antibiotic combinations were evaluated. The overall susceptibility rates of all isolates obtained by the Etest (ET) versus agar dilution (AD) were 60.4% versus 61.8% for penicillin, 63.8% versus 63.9% for erythromycin, 90.6% versus 96% for vancomycin, and 99.1% versus 96.0% for imipenem, respectively. Major discrepancies occurred in the testing of the susceptibility of Streptococcus mutans to vancomycin, with 59.5% (ET) versus 100% (AD), followed by S. salivarius, with 84.1% versus 100%; S. oralis, with 82.1% versus 96.4%; and S. mitis, with 90% versus 100%, respectively. There were also differences in the rates of susceptibility of S. mutans, 66.5% (ET) versus 85.1% (AD), and S. intermedius, 82.9% versus 72.1%, respectively, to penicillin. General agreement between the results of ET and AD was obtained for 973 organism-antibiotic combinations out of 1,080 antibiotic combinations, i.e., 90.1%. Very major errors were found for 6.8% of isolates, and major errors were found for 3.2% of isolates; the minor errors were negligible. Agreement between the results of the two methods was 98.7% for penicillin, 94.6% for vancomycin, 96.9% for imipenem, and 99.9% for erythromycin. The highest rate of very major errors was for vancomycin, at 5.4%. The ET appears to be as efficient as AD for susceptibility testing of VS, except for vancomycin, where very major errors in the results were relatively high.     

Impact of prolonged incubation on disk diffusion susceptibility test results for Staphylococcus aureus

Because strains of Staphylococcus aureus that are resistant to penicillinase-resistant penicillins may be difficult to detect in the clinical laboratory, a variety of changes in methodology have been suggested to increase their detection. In 1984, the West Los Angeles Veterans Administration Medical Center experienced an increase in clinically significant strains of oxacillin-resistant S. aureus. To insure that such strains would not be missed by the disk diffusion test methods employed for routine testing, changes in methodology were insituted. These included interpreting zone diameters around oxacillin disks at 48 h of incubation. We collected 139 isolates from patients thought to have oxacillin-resistant S. aureus based on these test results and later retested the isolates using microdilution MIC testing. Only 85 isolates (61%) had microdilution oxacillin MICs of greater than or equal to 8.0 micrograms/ml, whereas 54 (39%) had oxacillin MICs of less than or equal to 2.0 micrograms/ml. A review of medical records revealed that in 1 year there were 98 patients with isolates appearing resistant by disk diffusion but not confirmed by microdilution MICs; many patients were placed in isolation and treated with specific antimicrobial agents. We conclude that incubation of oxacillin disk diffusion tests for longer than 24 h in conjunction with disregard for resistance to other classes of antimicrobial agents may result in an unacceptably high degree of false resistance results. Because the resistance of S. aureus has important therapeutic and infection control implications, it is necessary to recognize problems that may result in ambiguous or inaccurate susceptibility results.