Comparison of Agar Dilution, Microdilution, E-Test, and Disk Diffusion Methods for Testing Activity of Cefditoren against Streptococcus pneumoniae

This study evaluated the susceptibility of pneumococci to cefditoren by agar dilution and microdilution methods (both in air) and by E-test (AB Biodisk, Solna, Sweden) and disk diffusion methods (both in CO(2)). By the three MIC tests, the MICs at which 50 and 90% of isolates were inhibited (MIC(50)s and MIC(90)s) were, respectively, as follows (in micrograms per milliliter): for the 65 penicillin-susceptible strains tested, 0.016 and 0.03 (by agar dilution), 0.016 and 0.03 (by microdilution), and 0.016 and 0.03 (by E test); for the 68 penicillin-intermediate strains tested, 0.125 and 0.5 (by agar dilution), 0.125 and 0.5 (by microdilution), and 0. 25 and 0.5 (by E test); and for the 67 penicillin-resistant strains tested, 1.0 and 1.0 (by agar dilution), 0.5 and 1.0 (by microdilution), and 1.0 and 1.0 (by E test). With tentative cefditoren breakpoints (in micrograms per milliliter) of /=8.0 (resistant), all strains were susceptible to cefditoren by agar, microdilution, and E-test results; with breakpoints of /=4.0 microg/ml, 97% of strains were cefditoren susceptible by agar dilution results, 98% were susceptible by microdilution results, and 99% were susceptible by E-test results. When microdilution and E-test results were compared to those from the reference agar dilution method, 191 (95.5%) and 183 (91.5%) of strains gave essential agreement (+/-1 log(2) dilution); 8 (2.7%) minor discrepancies were found for both methods with a breakpoint of /=20 (susceptible), 17 to 19 (intermediate), and /=16 mm (susceptible). All three methods for testing the MIC of cefditoren showed excellent correlation.     

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.     

Determination of Penicillin MICs for Streptococcus pneumoniae by Using a Two- or Three-Disk Diffusion Procedure

The potential for the use of the disk diffusion method to accurately predict penicillin MICs for Streptococcus pneumoniae was investigated with penicillin (6 μg), methicillin (5 μg), and oxacillin (1 μg) disks. A total of 183 S. pneumoniae isolates were tested by three MIC procedures (agar dilution, microdilution, and E-test). Regression analyses of the geometric mean of the three MIC results against (i) the sum of the zone diameters for methicillin, penicillin, and oxacillin disks; (ii) the sum of the zone diameters for methicillin and penicillin disks; and (iii) each of the three individual zone diameters were performed. Calculated MICs were determined from each of these regression analyses and compared to the mean reference MICs. A high level of correlation was obtained with both the two- and the three-disk procedures (r = 0.97), with essential agreement rates (±1 doubling dilution) between MICs calculated by the three-disk procedure and the two-disk procedure and the mean reference MICs of 98.4 and 98.9%, respectively. No major or very major errors were obtained with the two- or three-disk procedures. The accuracy of the disks used individually was lower (r = 0.84 to 0.93). However, oxacillin and methicillin disk testing remain excellent for screening strains, with all penicillin-susceptible strains having zones of >21 and >22 mm, respectively. The combination disk procedure, which involves the use of three disks (methicillin, oxacillin, and penicillin) or two disks (methicillin and penicillin) for testing S. pneumoniae, can provide accurate penicillin MICs and qualitative category results that are comparable to results obtained by the E-test, agar, and microdilution MIC methods.     

In vitro activity of ceftizoxime,ceftazidime, cefuroxime,ceftriaxone, cefotaxime,and penicillin againstStreptococcus pneumoniae as determined by three quantitative methods

E-test, broth microdilution, and agar dilution susceptibility tests were used to evaluate penicillin and five extended-spectrum cephalosporins against 196 strains ofStreptococcus pneumoniae with different levels of penicillin resistance. Oxacillin disk tests corresponded to minimum inhibitory concentrations of penicillin determined by the E-test better than those generated by broth microdilution or agar dilution methods. Relative potency of the study drugs was as follows: cefotaxime=ceftriaxone > penicillin > cefuroxime > ceftizoxime > ceftazidime.     

Evaluation of the E Test for Antimicrobial Susceptibility Testing of Pseudomonas aeruginosa Isolates from Patients with Long-Term Bladder Catheterization

The E test was evaluated in comparison with reference agar methods (National Committee for Clinical Laboratory Standards) for the susceptibility testing of 248 Pseudomonas aeruginosa isolates from bladder-catheterized patients against nine antibiotics. The E-test MICs correlated well with those determined by the agar dilution and disk diffusion reference methods (88 and 92.5% within 1 log₂ dilution step, respectively), confirming that the E test is a reliable method for the determination of MICs of antibiotics for catheterization-associated P. aeruginosa isolates.     

Activity of Gatifloxacin against Haemophilus influenzae and Moraxella catarrhalis, Including Susceptibility Test Development, E-Test Comparisons, and Quality Control Guidelines for H. influenzae

In vitro antimicrobial activity and susceptibility testing interpretation criteria and quality control were studied for gatifloxacin, a new 8-methoxy fluoroquinolone, tested against Haemophilus influenzae. Moraxella catarrhalis (600 strains) and H. influenzae (1,400 strains) from the SENTRY Antimicrobial Surveillance Program in North America (Canada and the United States) were also tested against gatifloxacin and 12 other antimicrobial agents. Gatifloxacin (MIC at which 90% of the isolates are inhibited [MIC90], /=18 mm) was also suggested for H. influenzae testing. No interpretive errors were observed. Quality control guidelines for H. influenzae ATCC 49247 were determined by using the NCCLS M23-T3 (1998) study design. The results from the nine-laboratory protocol suggested the following control ranges: for broth microdilution tests, 0.004 to 0.03 microg/ml; for disk diffusion testing, 33 to 41 mm. Gatifloxacin appears to be a potent anti-Haemophilus fluoroquinolone compound with in vitro testing interpretive criteria that will produce accurate results (disk diffusion, broth microdilution, and E-test).     

Correlation between microdilution, E-test, and disk diffusion methods for antifungal susceptibility testing of posaconazole against Candida spp

Agar-based antifungal susceptibility testing is an attractive alternative to the microdilution method. We examined the correlation between the microdilution, E-test, and disk diffusion methods for posaconazole against Candida spp. A total of 270 bloodstream isolates of Candida spp. with a broad range of posaconazole MICs were tested using the CLSI M27-A2 method for microdilution, as well as the M-44A method and E-test methods for agar-based testing on Mueller-Hinton agar supplemented with 2% glucose and 0.5 microg of methylene blue. MICs and inhibitory zone diameters at the prominent growth reduction endpoint were recorded at 24 and 48 h. The Candida isolates included Candida albicans (n = 124), C. parapsilosis (n = 44), C. tropicalis (n = 41), C. glabrata (n = 36), C. krusei (n = 20), C. lusitaniae (n = 3), and C. dubliniensis (n = 2). The overall concordance (i.e., the percentage of isolates within two dilutions) between the E-test and microdilution was 64.8% at 24 h and 82.6% at 48 h. When we considered an arbitrary breakpoint of < or = 1 microg/ml, the agreement between the E-test and microdilution methods was 87.8% at 24 h and 93.0% at 48 h. The correlation of MICs with disk diffusion zone diameters was better for the E-test than the microdilution method. Zone correlation for diameters produced by the disks of two manufacturers was high, with a Pearson test value of 0.941 at 24 h. The E-test and microdilution MICs show good concordance and interpretative agreement. The disk diffusion zone diameters are highly reproducible and correlate well with both the E-test and the microdilution method, making agar-based methods a viable alternative to microdilution for posaconazole susceptibility testing.     

Evaluating the resistance to posaconazole by E-test and CLSI broth microdilution methodologies of <Emphasis Type="Italic">Candida</Emphasis> spp. and pathogenic moulds

E-test methodology was compared with Clinical and Laboratory Standards Institute (CLSI) broth microdilution, particularly concerning the detection of resistance to posaconazole among clinical fungal isolates. The susceptibility of a large set of fungal strains (n = 300) was evaluated following 24 and 48 h in two different culture media (RPMI 1640 and Sabouraud agar). Fungal strains were highly susceptible to posaconazole; however, few less susceptible strains were found, mostly regarding Candida albicans, Candida glabrata, Acremonium sp., Cladosporium sp. and Scedosporium apiospermum. Broth microdilution and E-test methods provided similar results for posaconazole-susceptible strains, while the less susceptible fungal strains (10.3% of the strains showed MIC ≥2 μg/mL) resulted in higher discrepancies between the two methodologies, particularly concerning Candida spp. E-test susceptibility values were critically affected by the pH of the culture media. Sabouraud medium provided similar susceptibility results for moulds to those for RPMI, soon after 24 h. Posaconazole resistance was rare in this study, but routine susceptibility methods, such as the E-test, should be able to detect fungal strains with reduced susceptibility. E-test methodology still needs improvements to recognise accurately strains less susceptible to posaconazole.     

Extremely High Prevalence of Nasopharyngeal Carriage of Penicillin-Resistant Streptococcus pneumoniae among Children in Kaohsiung, Taiwan

Resistance (intermediate and high) to penicillin among Streptococcus pneumoniae strains is an emerging problem worldwide. From 1995 to 1997, isolates of S. pneumoniae not susceptible to penicillin were seen with increasing frequency from blood, cerebrospinal fluid, pleural fluid, and middle ear fluid from pediatric patients at the Veterans General Hospital-Kaohsiung. To determine the prevalence of carriage of these penicillin-nonsusceptible S. pneumoniae isolates, we obtained nasopharyngeal swab specimens from 2,905 children (ages, 2 months to 7 years) attending day-care centers or kindergartens or seen in our outpatient clinic. S. pneumoniae was isolated from 611 children, and 584 strains were available for analysis. The oxacillin disc test was used as a screening test to evaluate penicillin susceptibility. The MICs of 11 antibiotics (penicillin, cefaclor, cefuroxime, ceftriaxone, cefotaxime, imipenem, chloramphenicol, clarithromycin, rifampin, vancomycin, and teicoplanin) were determined by the E-test. Only 169 (29%) of the strains were susceptible to penicillin; 175 (30%) strains were intermediately resistant and 240 (41%) were highly resistant. The isolates also demonstrated high rates of resistance to other β-lactams (46% were resistant to cefaclor, 45% were resistant to cefuroxime, 45% were resistant to ceftriaxone, 31% were resistant to cefotaxime, and 46% were resistant to imipenem). The rate of resistance to macrolide antimicrobial agents was strikingly high; 95% of the isolates were not susceptible to clarithromycin. However, 97% were susceptible to rifampin and 100% were susceptible to the two glycopeptides (vancomycin and teicoplanin). While reports of penicillin-resistant S. pneumoniae increased worldwide through the 1980s, the high prevalence (71%) of resistance reported here is astonishing. Surveillance of nasopharyngeal swab specimen cultures may provide useful information on the prevalence of nonsusceptible strains causing invasive disease. Such information could be used to guide therapy of pneumococcal infections.     

Validation of 24-hour fluconazole MIC readings versus the CLSI 48-hour broth microdilution reference method: results from a global Candida antifungal surveillance program

We performed 24- and 48-h MIC determinations and disk diffusion testing of fluconazole against more than 11,000 clinical isolates of Candida species. By using the reference MIC breakpoints, the categorical agreement between the 24-h and reference 48-h broth microdilution results ranged from 93.8% (all Candida species) to 94.9% (all Candida species minus Candida krusei), with only 0.1% very major errors (VME). The essential agreement (within 2 log₂ dilutions) between the 24-h and 48-h results was 99.6%. The categorical agreement between the 24-h disk diffusion results and the 24-h MIC results, using the previously established breakpoints, was 94.4%, with 0.1% VME. Both the MIC and the disk diffusion results obtained for fluconazole after only 24 h of incubation may be used to determine the susceptibilities of Candida spp. to this widely used antifungal agent.     

Influence of variations in test methods on susceptibility of Haemophilus influenzae to ampicillin, azithromycin, clarithromycin, and telithromycin

The National Committee for Clinical Laboratory Standards standard broth microdilution method for testing the susceptibility of Haemophilus influenzae to ampicillin, azithromycin, clarithromycin, and telithromycin was evaluated by altering one variable at a time. Variables that were tested included age of colony for inoculum preparation, inoculum density, test medium, incubation atmosphere, and incubation time. For the macrolide, azalide, and ketolide agents, incubation in 5 to 7% CO(2) most significantly affected the MICs, producing nearly twofold increases for clarithromycin and telithromycin and a greater than threefold increase for azithromycin. For ampicillin, a 10-fold increase in inoculum density increased the geometric mean MICs for beta-lactamase-negative strains from 1. 50 to 2.45 microg/ml. In addition, 206 H. influenzae strains were tested for their susceptibilities to the same drugs by the broth microdilution tests in two media, as well as by agar dilution tests, disk diffusion tests, and Etests, on six different agar media. The three standard methods with Haemophilus test medium (HTM) compared favorably with each other except for a high minor discrepancy rate (27%) by the disk diffusion test with ampicillin and clarithromycin. Agar dilution test MICs on the five comparative media were generally higher than those on HTM agar but were only rarely more than one twofold concentration higher. Etest MICs of azithromycin and telithromycin were more than twofold higher than agar dilution and broth microdilution MICs on HTM; ampicillin Etest MICs were nearly twofold lower. The use of media other than HTM agar appears to have a minimal effect on susceptibility test results for the ketolide, azalide, or macrolide drugs that we tested against H. influenzae.     

Evaluation of in vitro spectra of activity of azithromycin, clarithromycin, and erythromycin tested against strains of Neisseria gonorrhoeae by reference agar dilution, disk diffusion, and Etest methods.

The macrolide-azilide susceptibility testing (agar dilution, disk diffusion, Etest) criteria for 105 Neisseria gonorrhoeae strains were evaluated. In addition, the potencies of azithromycin, clarithromycin, and erythromycin were studied. The most active macrolide-azilide agent was azithromycin (MIC at which 90% of the isolates are inhibited [MIC90], 0.5 microgram/ml) compared with clarithromycin (MIC90, 1.5 to 2 micrograms/ml) and erythromycin (MIC90, 2 to 4 micrograms/ml). The Etest (AB Biodisk, Solna, Sweden) was observed to produce MIC results very similar to those of the reference agar dilution test (GC agar base), with 100% of the results within 1 log2 dilution step of the reference MICs. The disk diffusion test zone diameters for all three drugs correlated at an acceptable level (r = -0.81 to -0.92) with the reference agar dilution MICs. Interpretive criteria for susceptibility were proposed for azithromycin at a MIC of < or = 2 micrograms/ml and a disk diffusion test zone of > or = 25 mm. No category for resistance was proposed because of the paucity of strains for which MICs were > 2 micrograms/ml. These tentative criteria should be further validated by correlations with clinical trial data for gonococcal strains (as they emerge) that have azithromycin MICs above the proposed susceptible category range.     

Antipneumococcal activity of telithromycin by agar dilution, microdilution, E test, and disk diffusion methodologies

Agar dilution and microdilution (both in air) and E test and disk diffusion (both in air and CO(2)) were used to test the activity of telithromycin against 110 erythromycin-susceptible and 106 erythromycin-resistant pneumococci. The MICs at which 50 and 90% of strains are inhibited (MIC(50)s and MIC(90)s, respectively) for erythromycin-susceptible strains varied between 0.008 and 0.016 microg/ml and 0.016 and 0.03 microg/ml when the samples were incubated in air. By comparison, telithromycin MIC(50)s and MIC(90)s for erythromycin-resistant strains were in air 0.03 to 0.125 and 0. 125 to 0.5 microg/ml, respectively. When agar dilution was used as the reference method, essential agreement was found for 112 of 216 strains (51.9%) for microdilution, 168 of 216 (77.8%) for E test in air, and 132 of 216 (61.1%) for E test in CO(2). With the exception of four strains tested by E test in CO(2), all organisms were susceptible to a proposed telithromycin susceptibility breakpoint of < or =1 microg/ml. By disk diffusion with 15-microg telithromycin disks, all strains but one had zones of inhibition > or =19 mm in diameter when incubated in CO(2), while all strains had zone diameters of > or = 22 mm when incubated in air. Zone diameters in air were generally 4 to 5 mm larger than in CO(2). By all methods, MICs and zones of all erythromycin-resistant strains occurred in clusters separated from those seen with erythromycin-susceptible strains. The results for macrolide-resistant strains with erm and mef resistance determinants were similar. The results show that (i) telithromycin is very active against erythromycin-susceptible and -resistant strains irrespective of macrolide resistance mechanism; (ii) susceptibility to telithromycin can be reliably tested by the agar, microdilution, E test, and disk diffusion methods; and (iii) incubation in CO(2) led to smaller zones by disk diffusion and higher MICs by E test, but at a susceptible MIC breakpoint of < or =1 microg/ml and a susceptible zone diameter cutoff of > or =19 mm in CO(2), 215 of 216 strains were found to be susceptible to telithromycin.     

Validation of diffusion methods for macrolide susceptibility testing of Helicobacter pylori

Helicobacter pylori resistance to macrolides is increasing, and the need for susceptibility testing has become crucial. The only standardized method is agar dilution, which is not adapted to clinical practice. The present work aimed: (1) to optimize the technical conditions and to assess the reproducibility of the E-test and disk diffusion method for macrolides susceptibility testing of H. pylori, and (2) to assess the performances of these two phenotypic methods in detecting strains harboring a resistance mechanism to macrolides. We used 191 isolates collected in nine centers of France and Belgium. Phenotypic tests were performed on Mueller-Hinton agar supplemented with 10% horse blood, inoculated with a 2-day-old H. pylori suspension (10(8) CFU/ml), and incubated for 72 hr at 37 degrees C under microaerophilic conditions. The reproducibility studied on two randomly selected strains was better for disk diffusion than for the E-test for both clarithromycin and erythromycin. For a subset of 10 strains, the MICs of erythromycin and clarithromycin did not differ from more than one two-fold dilution when determined by E-test or agar dilution method. The breakpoints were for MICs: 1 mg/L for both clarithromycin and erythromycin and for inhibition diameters, 22 mm for clarithromycin and 17 mm for erythromycin. There was a 100% concordance between susceptibility to erythromycin and clarithromycin. However, the susceptible and resistant populations were better separated by testing erythromycin. Of 34 resistant strains, two lacked the A2142G and A2143G point mutations in 23S rRNA by PCR-RFLP. None of 15 tested sensitive strains were positive for one of these two point mutations. For clinical practice, we recommend to assess macrolide susceptibility of H. pylori by using one of these two phenotypic methods under the described technical conditions.     

Comparison of Eight Methods To Detect Vancomycin Resistance in Enterococci

A collection of genetically unrelated vancomycin-resistant enterococci (VRE) including 50 vanA, 15 vanB, 50 vanC1, and 30 vanC2 VRE were used to evaluate the accuracy of eight currently available susceptibility test methods (agar dilution, disk diffusion, E-test, agar screen plate, Vitek GPS-TA and GPS-101, and MicroScan overnight and rapid panels). vanA VRE were detected by all methods. vanB VRE were often not detected by Vitek GPS-TA and MicroScan rapid (sensitivities, 47 and 53%, respectively), though the new Vitek GPS-101 was found to be a significant improvement. E-test and the agar screen were the only two methods detecting all VRE, including the vanC1/C2 VRE.     

Comparison of testing methods for detection of decreased linezolid susceptibility due to G2576T mutation of the 23S rRNA gene in Enterococcus faecium and Enterococcus faecalis

E-test, Vitek 2, MicroScan, agar dilution, and disk diffusion were compared for detection of decreased linezolid susceptibility due to 23S rRNA gene G2576T mutation among 32 clinical Enterococcus strains initially reported as intermediate or resistant by E-test alone or Vitek 2 confirmed by E-test. Agar and broth dilution methods were in concordance with PCR detection of the mutation, and disk diffusion was somewhat less sensitive but equally specific.     

ROLE OF AZITHROMYCIN AGAINST CLINICAL ISOLATES OF FAMILY ENTEROBACTERIACEAE: A COMPARISON OF ITS MINIMUM INHIBITORY CONCENTRATION BY THREE DIFFERENT METHODS

Purpose: To determine the effect of azithromycin, a new azalide antibiotic, on clinical isolates of the family Enterobacteriaceae and to determine and compare its minimum inhibitory concentration (MIC) by disk diffusion, agar dilution and E-test methods. Materials and Methods: One hundred fifty-nine bacterial strains belonging to the family Enterobacteriaceae, isolated from different clinical samples, were tested for their susceptibility to azithromycin by disk diffusion, agar dilution and E-test methods. The MIC values were analysed and the percentages of agreement between the different methods were mentioned. Results: Of the 159 isolates of the family Enterobacteriaceae, 60.37% were E. coli followed by Klebsiella species 28.3%, Salmonella and Shigella species 3.77% and Enterobacter and Citrobacter species 1.88% each. Maximum isolates were obtained from urine 117/159 (73.58%). Azithromycin was found to be more active against Salmonella and Shigella species, showing 100% sensitivity the by E-test and 83.33% by the disk diffusion methods. In the agar dilution method, 83.33% of Salmonella and 66.66% of Shigella species were sensitive to azithromycin. The overall agreement between disk diffusion and agar dilution method was 96.8%, between agar dilution and E-test was 88% and between disk diffusion and E-test was 91.2%. Conclusion: Azithromycin may become an important addition to our antimicrobial strategies, especially for the treatment of bacterial diarrhoea and infections caused by Salmonella typhi.     

Development of a Disk Diffusion Method for Testing Moraxella catarrhalis Susceptibility Using Clinical and Laboratory Standards Institute Methods: a SENTRY Antimicrobial Surveillance Program Report

Currently, there is no Clinical and Laboratory Standards Institute (CLSI) disk diffusion method for testing Moraxella catarrhalis susceptibility. We examined 318 clinical strains of M. catarrhalis obtained as part of the SENTRY (Asia-Pacific) Antimicrobial Surveillance Program, plus two ATCC strains. MICs were determined by the CLSI standard broth microdilution method, and zone diameters were determined on Mueller-Hinton agar incubated in 5% CO₂. All strains were examined for the presence of BRO-1 and BRO-2 β-lactamases by using molecular techniques. Tentative zone diameter interpretive criteria were successfully developed for 19 antimicrobial agents, including nine β-lactams, using current MIC interpretive criteria where available or wild-type cutoff values where no prior criteria were available. The proposed interpretive criteria were highly accurate, with ≤0.7% very major (falsely susceptible) and ≤1.0% major (falsely resistant) errors, respectively.Moraxella catarrhalis is a common commensal and occasionally pathogenic bacterium associated with a range of infections of the respiratory tract, including acute otitis media, acute sinusitis, and acute exacerbations of chronic bronchitis (5, 22, 34). Less commonly, this organism can cause more serious and invasive infection, including pneumonia, septicemia, and meningitis (4, 28). A number of standards-setting organizations, including the Société Française de Microbiologie (29) and the British Society for Antimicrobial Chemotherapy (http://www.bsac.org.uk/susceptibility_testing.cfm), have developed disk diffusion tests and interpretive criteria for M. catarrhalis. Recently, the Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing published an interpretive guideline (6), but only for broth microdilution tests, using criteria essentially the same as those applied to Haemophilus spp. (10). Routine testing was not recommended by CLSI at the time of development of those guidelines.However, this species is frequently isolated from respiratory tract infection specimens and, when present in large numbers, can be assumed by the laboratory to be playing a pathogenic or copathogenic role and, therefore, can be reported to the clinician. For the many international laboratories using CLSI methods and standards, the lack of a disk diffusion method and interpretive criteria limits their ability to communicate useful information to the clinician for a common potential pathogen. We followed CLSI M23-A3 guidelines (7) for the development of a disk diffusion test for M. catarrhalis, supplemented with molecular tests for some resistance mechanisms to assist in the future enhancement of interpretive criteria.(This work was presented in part at the European Congress of Clinical Microbiology and Infectious Diseases, Munich, Germany, 2007.)     

Metronidazole susceptibility testing for Helicobacter pylori: comparison of disk, broth, and agar dilution methods and their clinical relevance.

Since the methods for metronidazole susceptibility testing of Helicobacter pylori have not been standardized or validated, we compared three methods that are used to test the metronidazole susceptibilities of 25 isolates of H. pylori. Specifically, we examined the methods of Steer's replicator agar dilution, tube broth microdilution, and modified Kirby-Bauer disk diffusion. The metronidazole disk zone sizes obtained by the disk diffusion method correlated well (r = 0.74) with the MICs obtained by the agar dilution method. Afterward, the disk diffusion method was used to characterize the metronidazole susceptibilities of 44 isolates of H. pylori. Dual therapy (bismuth and metronidazole) proved to be highly effective against metronidazole-susceptible strains (81.6% eradication rate) but fared poorly against resistant strains (16.7% eradication rate; P < 0.01). Using agar dilution testing, we validated the modified Kirby-Bauer disk diffusion method for metronidazole susceptibility testing of H. pylori and conclude that it is practical, accurate, and clinically applicable.     

Comparative evaluation of the E test, agar dilution, and broth microdilution for testing susceptibilities of Helicobacter pylori strains to 20 antimicrobial agents.

The Epsilometer test (E test; AB Biodisk, Solna, Sweden), a new quantitative technique for the determination of antimicrobial susceptibility, was compared to reference methods (agar dilution and broth microdilution) for the antimicrobial susceptibility testing of Helicobacter pylori. Seventy-one H. pylori strains isolated from patients with duodenal ulcers were tested against 20 antimicrobial agents. The E test and the agar dilution method were carried out on Mueller-Hinton agar; the broth microdilution method was performed with Mueller-Hinton broth. The E-test results showed excellent correlation with the agar dilution results, with 91.3 and 98.8% agreement within 1 and 2 log2 dilution steps, respectively, in a total of 1,350 tests. The correlation between the E-test results and the broth microdilution results was slightly higher, with 91.6 and 99.1% agreement within 1 and 2 log2 dilution steps, respectively, in a total of 1,317 tests. There were six major errors and two very major errors by the metronidazole E test compared to the results obtained by reference methods. Excellent agreement between E-test, agar dilution, and broth microdilution results was found for resistance to erythromycin (8%), clarithromycin (6%), and tetracycline (6%). Our results confirm that the E test is comparable to standardized methods for susceptibility testing. Therefore, the E test is a reliable and alternative method for testing H. pylori susceptibility to a wide range of antimicrobial agents in clinical practice.