Comparison of disk diffusion, VITEK 2, and broth microdilution antimicrobial susceptibility test results for unusual species of Enterobacteriaceae

We compared the antimicrobial susceptibility testing results generated by disk diffusion and the VITEK 2 automated system with the results of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) reference method for 61 isolates of unusual species of Enterobacteriaceae. The isolates represented 15 genera and 26 different species, including Buttiauxella, Cedecea, Kluyvera, Leminorella, and Yokenella. Antimicrobial agents included aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillins, and trimethoprim-sulfamethoxazole. CLSI interpretative criteria for Enterobacteriaceae were used. Of the 12 drugs tested by BMD and disk diffusion, 10 showed >95% categorical agreement (CA). CA was lower for ampicillin (80.3%) and cefazolin (77.0%). There were 3 very major errors (all with cefazolin), 1 major error (also with cefazolin), and 26 minor errors. Of the 40 isolates (representing 12 species) that could be identified with the VITEK 2 database, 36 were identified correctly to species level, 1 was identified to genus level only, and 3 were reported as unidentified. VITEK 2 generated MIC results for 42 (68.8%) of 61 isolates, but categorical interpretations (susceptible, intermediate, and resistant) were provided for only 22. For the 17 drugs tested by both BMD and VITEK 2, essential agreement ranged from 80.9 to 100% and CA ranged from 68.2% (ampicillin) to 100%; thirteen drugs exhibited 100% CA. In summary, disk diffusion provides a reliable alternative to BMD for testing of unusual Enterobacteriaceae, some of which cannot be tested, or produce incorrect results, by automated methods.     

Use of Haemophilus test medium for broth microdilution antimicrobial susceptibility testing of Streptococcus pneumoniae.

A recently described medium (Haemophilus test medium [HTM]) for antimicrobial susceptibility testing of Haemophilus influenzae was evaluated in this study for broth microdilution testing of Streptococcus pneumoniae. A total of 137 clinical isolates was tested against 11 antimicrobial agents, using Mueller-Hinton broth supplemented with 3% lysed horse blood in parallel with HTM. Inocula of 5 X 10(5) CFU/ml and incubation for 20 to 24 h were used with both media. All isolates of S. pneumoniae produced acceptable growth in both media, and MICs determined in HTM agreed closely with those determined in lysed horse blood. Drugs which provided a MIC within 1 log2 concentration difference in both media included penicillin (100%), ampicillin (98.0%), amoxicillin-clavulanate (100%), ampicillin-sulbactam (100%), cephalexin (98.9%), cefaclor (96.8%), cefuroxime (99.0%), chloramphenicol (96.2%), tetracycline (96.2%), and erythromycin (100%). HTM MICs with trimethoprim-sulfamethoxazole were 1 to 2 log2 concentration increments higher in 92.0% of isolates than MICs determined in lysed horse blood. Based on the results of this study, HTM appears to represent a promising alternative medium for broth microdilution susceptibility testing of S. pneumoniae.     

Comparison of broth macrodilution, broth microdilution, and E test antifungal susceptibility tests for fluconazole.

A comparison of the E test, the broth microdilution test, and the reference broth macrodilution susceptibility test of the National Committee for Clinical Laboratory Standards for fluconazole susceptibility testing was performed with 238 clinical isolates of Candida species and Torulopsis (Candida) glabrata. An 80% inhibition endpoint MIC was determined by the reference broth macrodilution method after 48 h of incubation. The MICs obtained by the two study methods were read after 24 and 48 h of incubation. Overall, excellent agreement within 2 doubling dilutions was obtained between the broth microdilution and the broth macrodilution methods for the combined results for all species at both 24 h (93%) and 48 h (94%). The correlation of 24-h MIC endpoints between the E test and the broth macrodilution methods was 37% for T. glabrata, 56% for Candida tropicalis, 93% for Candida albicans, and 90% for other Candida species. The percent agreement at 48 h ranged from 34% for T. glabrata to 97% for Candida species other than C. albicans and C. tropicalis. These initial results support the further evaluation of the E test as an alternative method for fluconazole susceptibility testing of Candida species.     

Comparison of Etest method with reference broth microdilution method for antimicrobial susceptibility testing of Yersinia pestis

The utility of Etest for antimicrobial susceptibility testing of Yersinia pestis was evaluated in comparison with broth microdilution and disk diffusion for eight agents. Four laboratories tested 26 diverse strains and found Etest to be reliable for testing antimicrobial agents used to treat Y. pestis, except for chloramphenicol and trimethoprim-sulfamethoxazole. Disk diffusion testing is not recommended.     

Validation of commercial dry-form broth microdilution panels for susceptibility testing of AZD2563, a new long-acting oxazolidinone

The MIC results using a dry-form broth microdilution panel (TREK Diagnostic/Sensititre, Westlake, OH, USA) were validated for AZD2563, a novel oxazolidinone compound. In comparision studies against reference frozen-form panels, the commercial MIC results were the same as the reference calue for 82.7% of organisms and all results were within ± one log₂ dilution. Using 462 organisms, most from three genus groups (enterococci, staphylococci, streptococci), test results indicate that Sensititre MIC values were comparable to the reference test and can be utilized in clinical trials of for routine laboratory use when testing AZD2563 and linezolid, the drug class comparator.     

Comparison of agar disk diffusion, microdilution broth, and agar dilution for testing antimicrobial susceptibility of coagulase-negative staphylococci.

A collection of 120 oxacillin-susceptible and 120 oxacillin-resistant coagulase-negative staphylococci (CNS) from six tertiary care hospital laboratories were tested by agar disk diffusion, three microdilution broth systems (Sensititre, Dynatech, and Alpkem), and the Vitek AutoMicrobic system for comparison with reference agar dilution results. The antimicrobial agents tested were oxacillin, cefazolin, cefotaxime, cefuroxime, cefamandole, fusidic acid, rifampin, and vancomycin. Incubation was at 30 or 35 degrees C for 24, 48, and 72 h. The broth media were supplemented with 2% NaCl for some antimicrobial agents, and the agar dilution method was used with and without the addition of 4% NaCl. The CNS were identified to species by the method of Kloos and Schleifer. The results showed a lack of concordance between two hospitals with respect to oxacillin susceptibility testing by agar dilution with no NaCl supplement. The reasons are not clear but may be related to variations in media. The 4% NaCl supplement or extended incubation to 48 h eliminated this difference. The cefazolin and cefotaxime susceptibility results in the agar disk diffusion test were unreliable if accepted at face value. Cefamandole testing correlated well with the reference method regardless of the method used, and salt supplementation is not recommended. Most of the oxacillin-resistant CNS were resistant to the other beta-lactam drugs except cefamandole. Of 22 CNS resistant to cefamandole, 21 were S. haemolyticus.     

Comparison of BIOGRAM and commercial microdilution antimicrobial susceptibility test systems.

The BIOGRAM (Difco Laboratories, Detroit, Mich.) system, which is designed to calculate MICs from disk diffusion zone diameters, was compared with two commercial microdilution antimicrobial susceptibility systems. A total of 111 clinical isolates were evaluated with each test system. Six additional isolates were tested in a comparison between BIOGRAM and Sceptor (Johnston Laboratories, Inc. Towson, Md.) systems. BIOGRAM demonstrated an overall correlation with the Sceptor microdilution method of 95.7% for 1,287 organism-antimicrobial susceptibility combinations. The BIOGRAM and UniScept (Analytab Products, Inc., Plainview, N.Y.) systems were in agreement in 90.3% of 1,048 organism-antimicrobial susceptibility combinations tested. All methicillin-resistant staphylococci were detected by the standard disk agar diffusion method used with the BIOGRAM system. The BIOGRAM system provides an acceptable alternative to these commercial systems for the determination of quantitative susceptibility.     

Quality control guidelines for disk diffusion and broth microdilution antimicrobial susceptibility tests with seven drugs for veterinary applications

This multicenter study proposes antimicrobial susceptibility (MIC and disk diffusion methods) quality control (QC) parameters for seven compounds utilized in veterinary health. Alexomycin, apramycin, tiamulin, tilmicosin, and tylosin were tested by broth microdilution against various National Committee for Clinical Laboratory Standards (NCCLS)-recommended QC organisms (Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853). In addition, disk diffusion zone diameter QC limits were determined for apramycin, enrofloxacin, and premafloxacin by using E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. aureus ATCC 25923. The results from five or six participating laboratories produced >/=99.0% of MICs and >/=95.0% of the zone diameters within suggested guidelines. The NCCLS Subcommittee for Veterinary Antimicrobial Susceptibility Testing has recently approved these ranges for publication in the next M31 document.     

Selection of a surrogate agent (vancomycin or teicoplanin) for initial susceptibility testing of dalbavancin: results from an international antimicrobial surveillance program

The immediate lack of market-dominating commercial products (Vitek or MicroScan) for susceptibility testing of the new glycolipopeptide, dalbavancin, requires a surrogate marker agent to assist microbiologists in the correct categorization of potentially indicated species (staphylococci and streptococci). Error-rate analyses for 16,749 isolates using vancomycin or teicoplanin results to categorize dalbavancin susceptibilities demonstrated that both glycopeptide agents were highly predictive of dalbavancin-susceptible results (nearly 100%) with only a rare minor error. Vancomycin test results most reliably predict dalbavancin susceptibility until validated commercial reagents become available for direct testing in clinical practice.     

Discordant results between the broth disk elution and broth microdilution susceptibility tests with Bacteroides fragilis group isolates.

Susceptibility testing of 161 clinical isolates of the Bacteroides fragilis group was performed to compare interpretive results generated by the broth disk elution and broth microdilution methods recommended by the National Committee for Clinical Laboratory Standards. Among the cephalosporin-cephamycin compounds tested, correlation was poorest for ceftizoxime (71%), ceftriaxone (57%), and cefotaxime (47%); when the tests did not correlate, false resistance was seen 92, 95, and 93% of the time, respectively. Cefotetan and cefoperazone showed lack of correlation in 19 and 20% of the tests, respectively. For cefotetan, false resistance was more frequent, while with cefoperazone, false susceptibility occurred more often. Cefoxitin produced the fewest discrepancies; 10% of the disk elution tests produced either false-resistance or false-susceptibility results. Mezlocillin and piperacillin showed lack of correlation in 8 and 14% of the tests, respectively, and discrepancies were due primarily to false-resistance results. Overall with the beta-lactams, 84% of the discordant interpretive results were false resistance by the broth disk elution test. Clindamycin had a discrepancy rate of 10%, with the majority of discrepancies being false susceptibility disk elution results. Because of the high number of discrepancies noted with ceftizoxime, ceftriaxone, and cefotaxime, we recommend that these drugs not be tested by the disk elution method and that they be tested by a quantitative MIC method such as the broth microdilution test. Furthermore, caution should be exercised when interpreting broth disk elution results with all the beta-lactams included in this study except imipenem. These data indicate the lack of correlation of results between these two tests for many beta-lactams and suggest the need for a reexamination of the disk elution method to provide a more accurately standardized test.     

Evaluation of broth microdilution susceptibility results for anaerobic organisms by use of a rapid direct colony inoculum.

A direct colony inoculum suspension procedure was compared with the overnight suspension procedure recommended for the broth microdilution anaerobic commercial system (Micro-Media Systems, Inc., Potomac, Md.). Six National Committee for Clinical Laboratory Standards-recommended quality control organisms, Bacteroides fragilis ATCC 25285, Clostridium perfringens ATCC 13124, Bacteroides thetaiotaomicron ATCC 29741, Bacteroides vulgatus ATCC 29327, Peptococcus magnus ATCC 29328, Peptococcus asaccharolyticus ATCC 29743, and 50 anaerobic clinical isolates were tested against seven commonly tested antimicrobial agents. The minimum inhibitory concentration results from each suspension method (using the quality control organisms) were identical in 18 (78%) instances, and within +/- 1 log2 dilution in 96% of the comparisons. Results with the fresh clinical isolates also compared satisfactorily with the overnight procedure (97% were identical or within one dilution). The Wilkins-Chalgren test medium failed to support the growth of most anaerobic gram-positive cocci and Bacteroides melaninogenicus strains.     

Disk diffusion susceptibility testing and broth microdilution quality control guidelines for BMY-28100, a new orally administered cephalosporin.

The BMY-28100 30-micrograms-disk test was evaluated by using 615 clinical isolates. Regression analyses and error rates were determined, leading to the recommendation of greater than or equal to 18-mm zone diameters (MIC correlate, greater than or equal to 8.0 micrograms/ml) for susceptibility and less than or equal to 14-mm zone diameters (MIC correlate, greater than or equal to 32 micrograms/ml) for resistance. Nearly all false-susceptible disk test results were among the Providencia spp. and the beta-lactamase-positive Haemophilus influenzae strains. Susceptibility disk test results for these species should be interpreted with caution. The following broth microdilution MIC quality control guidelines were determined from results of a multilaboratory trial: Escherichia coli ATCC 25922, 1.0 to 4.0 micrograms/ml; Enterococcus faecalis ATCC 29212, 4.0 to 16 micrograms/ml; Staphylococcus aureus ATCC 29213, 0.25 to 1.0 microgram/ml; and Pseudomonas aeruginosa ATCC 27853, greater than 32 micrograms/ml.     

Evaluation of broth microdilution antifungal susceptibility testing conditions for Trichophyton rubrum

Fifty clinical isolates of Trichophyton rubrum were selected to test with ketoconazole, fluconazole, itraconazole, griseofulvin, and terbinafine by following the National Committee for Clinical Laboratory Standards susceptibility testing guidelines for filamentous fungi (M38-A). In addition, other susceptibility testing conditions were evaluated: (i) three medium formulations including RPMI 1640 (standard medium), McVeigh & Morton (MVM), and Sabouraud dextrose broth (SDB); (ii) two incubation temperatures (28 and 35 degrees C); and (iii) three incubation periods (4, 7, and 10 days). The strains Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), T. rubrum (ATCC 40051), and Trichophyton mentagrophytes (ATCC 40004) were included as quality controls. All isolates produced clearly detectable growth only after 7 days of incubation. MICs were significantly independent of the incubation temperature (28 or 35 degrees C) (P < 0.05). Different incubation periods resulted in MICs which were consistently different for each medium when azoles and griseofulvin were tested (P < 0.05). MICs obtained from different media at the same incubation time for the same isolate were significantly different when azoles and griseofulvin were tested (P < 0.05). MICs were consistently higher (usually 1 to 2 dilutions) with RPMI than with MVM or SDB (P < 0.05). When terbinafine was tested, no parameter had any influence on MICs (P < 0.05). RPMI standard medium appears to be a suitable testing medium for determining the MICs for T. rubrum. MICs obtained at different incubation times need to be correlated with clinical outcome to demonstrate which time has better reliability.     

Comparison of disk diffusion,MIC test strip and broth microdilution methods for cefiderocol susceptibility testing on carbapenem-resistant enterobacterales

ObjectivesCefiderocol is a catechol-substituted cephalosporin dedicated to the treatment of infections caused by multidrug resistant gram-negative rods. Cefiderocol susceptibility testing might be complex. We compared cefiderocol susceptibility testing methods on a relevant collection of carbapenem-resistant Enterobacterales.MethodsCE-IVD (European CE marking required for all in vitro diagnostic (IVD)) broth microdilution (BMD) plate (ThermoFisher, Waltham, MA, USA) using regular Mueller-Hinton broth, MIC test strip (Liofilchem, Teramo, Italy), and disk diffusion (Liofilchem) were compared to a frozen BMD plate prepared with iron depleted Mueller-Hinton broth. First, a collection of 100 entirely sequenced carbapenem-resistant Enterobacterales was used to compare these methods. Then, a prospective comparison of disk diffusion and CE-IVD BMD was performed on 827 consecutive carbapenem non-susceptible Enterobacterales including 634 carbapenemase-producers.ResultsCompared to reference method, CE-IVD BMD plate gave 95.0% (95% CI, 88.8–97.9) categorisation agreement (CA), 2.8% (95% CI, 0.4–14.2) very major errors (VME), and 1.6% (95% CI, 0.3–8.7) major errors (ME) with high reproducibility. MIC strip gave only 63% (95% CI, 53.2–71.8) of CA and 94.9% (95% CI, 83.1–98.6) of VME due to critical underestimation of the MICs. Disk diffusion gave 77% (95% CI, 67.9–84.2) CA with additional 8% of the isolates within the area of technical uncertainty of 18–22 mm.Prospectively, disk diffusion gave 81.7% (95% CI, 79.0–84.2) CA, 23.3% (95% CI, 15.1–34.2%)VME, and 4.9% (95% CI, 3.6–6.7) ME. Additionally, 21.3% (95% CI, 18.6–24.2) of CRE were within the area of technical uncertainty.DiscussionCommercial CE-IVD BMD (ThermoFisher) is accurate for cefiderocol MIC determination in difficult-to-treat Enterobacterales whereas MIC test strip (Liofilchem), that was formulated for Pseudomonas aeruginosa only, should be avoided. Disk diffusion might be useful for screening, but many of these CRE have to be re-tested using BMD to assess definitive categorization.     

药敏试验E test法与琼脂扩散法的方法学比较

介绍一种新的药敏试验Ｅｔｅｓｔ（ＡＢＢｉｏｄｉｓｋ，Ｓｏｌｎａ，Ｓｗｅｄｅｎ）法，并与常用的纸片扩散法进行比较。Ｅｔｅｓｔ法系利用琼脂扩散法的原理，把一载有抗生素连续指数梯度浓度的５×５０ｍｍ的塑料条放于已接种菌液的琼脂板上，此条包含１５个ｌｏｇ２的稀释倍数，从抑菌环与载体条交界处可定量读出ＭＩＣ值。分别用Ｅｔｅｓｔ和纸片扩散法测定了临床分离的３１０株革兰氏阴性杆菌对１０种抗生素的敏感性。结果显示Ｅｔｅｓｔ与纸片扩散法的一致性达９０．１％，相关性在０．７５～０．９３之间，平均为０．８７，以Ｅｔｅｓｔ法为标准计算纸片扩散法的平均极大误差为１．５％，大误差为２．６％。结论认为Ｅｔｅｓｔ是一种精确可靠的药敏方法；常规纸片扩散法必须严格按照标准化进行。     

Evaluation of CLSI agar dilution method and Trek Sensititre broth microdilution panel for determining antimicrobial susceptibility of Streptococcus pneumoniae

Both the CLSI agar dilution method and Trek Sensititre broth microdilution panel for Streptococcus pneumoniae antimicrobial susceptibility testing were evaluated against the reference CLSI broth microdilution method using the most recently published CLSI breakpoints. While agar dilution was not an optimal method, the commercial panel appeared to be an acceptable method, with minor errors encountered for ceftriaxone, penicillin, and meropenem.     

Evaluation of the PASCO strep plus broth microdilution antimicrobial susceptibility panels for testing Streptococcus pneumoniae and other Streptococcal species

Antimicrobial resistance continues to increase worldwide among isolates of Streptococcus pneumoniae and other species of streptococci. Increasing rates of penicillin resistance, particularly in viridans group streptococci, and resistance to multiple classes of antimicrobial agents, including beta-lactams, macrolides, and fluoroquinolones, in pneumococci have increased the importance of having accurate antimicrobial susceptibility testing results for guiding therapy. One commercial method of assessing resistance in streptococci is the PASCO Strep Plus panel. This broth microdilution-based method has recently been expanded to include a variety of newer antimicrobial agents. Therefore, we compared the results of the new PASCO Strep Plus panels for 26 antimicrobial agents against the results generated using the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution reference method for 75 pneumococci and 68 other streptococcal isolates. Only 4 (0.2%) very major errors (all with pneumococci and each with a different antimicrobial agent) were observed. There were 5 (0.3%) major errors observed with pneumococci (each with a different antimicrobial agent), but only 1 major error with nonpneumococcal streptococci. All of the very major and major errors resolved on retesting. Of the 65 (3.9%) and 17 (1.6%) minor errors observed with pneumococci and other streptococci, respectively, all were within 1 dilution of the broth microdilution reference MIC result. Thus, the PASCO Strep Plus panel has comparable accuracy to the NCCLS broth microdilution reference method.     

Multicenter evaluation of the use of Haemophilus test medium for broth microdilution antimicrobial susceptibility testing of Streptococcus pneumoniae and development of quality control limits.

A five-laboratory collaborative study was undertaken to determine the precision and accuracy of broth microdilution susceptibility tests of Streptococcus pneumoniae isolates performed with Haemophilus test medium (HTM) compared with tests performed with lysed horse blood-supplemented Mueller-Hinton broth (LHB). The intra-and interlaboratory reproducibilities of MICs of 10 antimicrobial agents determined with the two media were found to be quite similar and highly reproducible in both media. On the basis of favorable performance in this study, S. pneumoniae ATCC 49619 is recommended as a quality control strain to assess the performance of HTM when this medium is used for testing of pneumococci. Testing of 293 unique clinical isolates of S. pneumoniae with both media in the respective participant laboratories allowed a direct comparison of MIC results and a calculation of interpretive error rates. Although there were some slight differences between MICs determined with HTM and MICs determined with LHB, few very major or major errors resulted from testing the clinical isolates against the 10 antimicrobial agents. However, MIC-interpretive criteria specific for S. pneumoniae should be developed and promulgated through a national consensus mechanism.     

Quality control limits for broth microdilution susceptibility tests of ten antifungal agents

Broth microdilution susceptibility tests of Candida species have now been standardized by the National Committee for Clinical Laboratory Standards (NCCLS). An eight-laboratory collaborative study was carried out in order to document reproducibility of tests of Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 by the NCCLS method. Replicate broth microdilution tests were used to define control limits for 24- and 48-h MICs of amphotericin B, flucytosine, fluconazole, voriconazole, ketoconazole, itraconazole, caspofungin (MK 0991), ravuconazole (BMS 207147), posaconazole (SCH 56592), and LY 303366.