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.     

Inter-technique variability between antimicrobial susceptibility testing methods affects clinical classification of cefuroxime in strains close to breakpoint

ObjectivesThe aim of this study was to evaluate the accuracy of various susceptibility methods when testing cefuroxime against a collection of Escherichia coli isolates with MIC values close to the breakpoint.Methods80 E. coli strains with a cefuroxime MIC value of 16 mg/L obtained by broth microdilution with Vitek 2 were selected. Microdilution was considered the reference standard and was performed in duplicate, as were disc and gradient diffusion tests using two different manufacturers in each case. EUCAST 8.0 breakpoints were used for MIC interpretation.ResultsAll strains were resistant according to Vitek 2 (MIC 16 mg/L) but 72.5% (58/80) were classified as susceptible by reference standard microdilution. Categorical and essential agreements between Vitek 2 and reference standard microdilution were 27.5% (95% CI 1.9–1.4) and 86.3% (95% CI 0.8–0.9), respectively. Differences are statistically significant when isolates are classified as ‘susceptible’ or ‘resistant’ according to EUCAST breakpoints between diffusion methods (disc and gradient) and reference standard microdilution. Using BioMérieux (BM) and Liofilchem (LF) gradient testing, 24.1% (14/58) and 13.8% (8/58) of results were identified as false susceptible and 4.5% (1/22) and 40.9% (9/22) were found to be false resistant, respectively. Using Oxoid (OX) and Bio Rad (BR) cefuroxime discs, 22.5% (13/58) and 17.2% (10/58) of results were false susceptible and 9.1% (2/22) and 13.6% (3/22) were false resistant, respectively.DiscussionIntertechnique variation around the cefuroxime breakpoint was a considerable source of disagreements and seriously affected the clinical classification of the isolates. We propose that the definition of the area of technical uncertainty (ATU) be modified to include the variability between approved AST methods.     

Comparison of five methods, including the PDM Epsilometer test (E test), for antimicrobial susceptibility testing of Pseudomonas aeruginosa.

The antimicrobial susceptibilities of 100 clinical isolates of Pseudomonas aeruginosa to six antipseudomonal antibiotics were tested by five methods: the National Committee for Clinical Laboratory Standards (NCCLS) methods for broth microdilution, agar dilution, and agar disk diffusion; the Vitek Automicrobic System method (Vitek Systems, Hazelwood, Mo.); and the PDM Epsilometer test (E test) (AB Biodisk, Solna, Sweden). The E test results showed excellent correlation with agar dilution results, with over 90% agreement within 1 doubling dilution between the E test and reference agar dilution MICs for all antimicrobial agents tested. The E test results also showed good correlation with the results from the reference agar disk diffusion method, with 90 to 99% complete agreement and 100% essential agreement on categories for all antibiotics tested (essential agreement is the agreement obtained when minor discrepancies are ignored). Comparison of categories with the E test and broth microdilution methods, using the broth microdilution method as the reference method, gave only 59% complete agreement for gentamicin, with 28 minor discrepancies and 13 very major discrepancies. Some discrepancies were observed between results from the E test and broth methods for gentamicin, with the broth microdilution and Vitek methods giving higher MICs than the E test and other methods using agar. The most recent NCCLS guidelines for broth dilution testing have reduced the recommended levels of cation supplementation, which may enhance future agreement between results for the aminoglycosides and P. aeruginosa on broth and on agar. We found that the E test offers a simple, labor-efficient, and accurate method for MIC determination on an agar medium.     

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.     

Comparison of the E Test to agar dilution, broth microdilution, and agar diffusion susceptibility testing techniques by using a special challenge set of bacteria.

The E Test (AB Biodisk, Solna, Sweden) is a new method for performing antimicrobial susceptibility tests. It consists of an impervious carrier (5- by 50-mm strip) with a predefined antimicrobic gradient which is placed on an inoculated agar plate and processed like a disk diffusion test. Results are generated directly as MICs from a continuous concentration gradient covering 15 twofold dilutions, and MICs are read where the edge of the inhibition zone intersects the strip. We compared the E Test with disk diffusion, broth microdilution, and agar dilution tests by using a challenge set of 195 gram-positive and gram-negative bacteria for 14 antimicrobial agents. Also, disk diffusion, broth microdilution, and agar dilution tests were compared with each other. All test method comparisons gave greater than 94% agreement for the category of susceptibility. The E Test category agreement with disk diffusion and broth microdilution was 95.1%, and with agar dilution it was 95.2%. The E Test results were as reliable as the results obtained by the standard antimicrobial susceptibility testing methods.     

Antimicrobial susceptibility testing of colistin – evaluation of seven commercial MIC products against standard broth microdilution for Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa,and Acinetobacter spp.

ObjectiveBoth EUCAST and CLSI recommend broth microdilution (BMD) for antimicrobial susceptibility testing of colistin, but BMD is rarely used in routine microbiology laboratories. The objective of this study was to evaluate five commercially available BMD products and two brands of gradient tests for colistin MIC determination using BMD according to ISO standard 20776-1 as reference.MethodsColistin MIC determination was performed according to the manufacturer's instructions on five commercially available BMD products (Sensititre, MICRONAUT-S, MICRONAUT MIC-Strip, SensiTest, and UMIC) and two gradient tests (Etest and MIC Test Strip). Colistin reference MICs were determined using frozen panels according to ISO standard 20776-1. An international collection of Gram-negative bacteria (n=75) with varying levels of colistin susceptibility was tested.ResultsThe colistin BMD products correlated well with reference tests, in particular for Sensititre and the two MICRONAUT products (essential agreement ≥96%: 66/69 (96%, CI 88–99%), 72/75 (96%, CI 88–99%) and 74/75 (99%, CI 92–100%)). The results were somewhat poorer for the BMD products SensiTest and UMIC: EA 88% (51/58, CI 77–95%) and 82% (61/74, CI 72–89%), respectively), and considerably poorer for the gradient tests (EA 43–71% depending on gradient test and Mueller-Hinton agar manufacturer). The gradient tests generally underestimated colistin MICs, resulting in a significant number of false susceptible results (9–18 of total 75 tests, compared with 1–3 for the BMD products).ConclusionsBased on the results of this study, we advise laboratories not to trust gradient tests for colistin susceptibility testing and to use broth microdilution methods for this purpose. There are several commercial broth microdilution tests available and in principle they perform well.     

Comparison of broth microdilution,E Test,and agar dilution methods for antibiotic susceptibility testing of Campylobacter jejuni and Campylobacter coli

A standardized broth microdilution method was compared to the E test and an agar dilution method for the antimicrobial susceptibility testing of Campylobacter jejuni and C. coli isolates. A group of 47 human clinical isolates, 37 isolates from retail poultry, and 29 isolates from living turkeys (total, 113 isolates) was included in the study. These encompassed 92 C. jejuni and 21 C. coli strains. The MICs of six antimicrobial agents were determined by the broth microdilution and E test methods, and the strains of human origin were additionally tested by the agar dilution method. In general, broth microdilution MICs agreed within 1 log(2) MIC increment with 90.0% of E test results and 78.7% of agar dilution test results. The agar dilution method gave much lower gentamicin MICs than the broth microdilution method, but the data were significantly (P < 0.01) correlated and there was 100% agreement in the sensitivities and specificities in the comparison of the tests. The broth microdilution method had the highest sensitivity for analysis of the susceptibilities of Campylobacter to nalidixic acid and trimethoprim-sulfamethoxazole. The MICs of ciprofloxacin and erythromycin complied numerically by all three methods. The classification of the results and the correlation of the data demonstrated a high degree of agreement. All methods were equally suitable for the testing of the sensitivity of Campylobacter to tetracycline. Thus, the broth microdilution method appears to be an easy and reliable method for determination of the MICs of antibiotics for C. jejuni and C. coli, and it may offer an interesting alternative to MIC determination by the agar dilution technique or the E test.     

Colistin MIC Variability by Method for Contemporary Clinical Isolates of Multidrug-Resistant Gram-Negative Bacilli

In vitro evaluation of colistin susceptibility is fraught with complications, due in part to the inherent cationic properties of colistin. In addition, no reference method has been defined against which to compare the results of colistin susceptibility testing. This study systematically evaluated the available methods for colistin MIC testing in two phases. In phase I, colistin MICs were determined in 107 fresh clinical isolates of multidrug-resistant (MDR) Gram-negative bacilli (GNB) by broth microdilution with polysorbate 80 (BMD-T), broth macrodilution (TDS), and the Etest. In phase II, 50 of these isolates, 10 of which were colistin resistant, were tested in parallel using BMD-T, TDS, agar dilution, broth microdilution without polysorbate 80 (BMD), and the TREK Gram-negative extra MIC format (GNXF) Sensititre. The Etest was also performed on these 50 isolates using Mueller-Hinton agar (MHA) from three different manufacturers. Colistin MIC results obtained from the five methods were compared to the MIC results obtained using BMD-T, the method that enables the highest nominal concentration of colistin in the test medium. Essential agreement ranged from 34% (BMD) to 83% (TDS), whereas categorical agreement was >90% for all methods except for BMD, which was 88%. Very major errors (VMEs) (i.e., false susceptibility) for the Etest were found in 47 to 53% of the resistant isolates, depending on the manufacturer of the MHA that was used. In contrast, VMEs were found for 10% (n = 1) of the resistant isolates by BMD and 0% of the isolates by the TDS, agar dilution, and Sensititre methods. Based on these data, we urge clinical laboratories to be aware of the variable results that can occur when using different methods for colistin MIC testing and, in particular, to use caution with the Etest.     

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.     

Wide variability in Pseudomonas aeruginosa aminoglycoside results among seven susceptibility testing procedures.

Seven commonly used antimicrobial susceptibility testing methods were used to test the susceptibility of 150 isolates of Pseudomonas aeruginosa against gentamicin, tobramycin, amikacin, carbenicillin, and piperacillin. Results were compared with respect to the susceptibility characteristics of the population of isolates as defined by each method. Conventional methods included agar disk diffusion and agar dilution, carried out in accordance with current recommendations of the National Committee for Clinical Laboratory Standards, as well as broth microdilution testing with cation-supplemented Mueller-Hinton broth (CSMHB). Methods in which instrumentation was used for result determination included the Autobac I, Avantage, Sensititre Autoreader (using a breakpoint panel at 18 h of incubation), and Vitek (AMS-240, using the GNS susceptibility card). When necessary for comparison, MIC data were converted to categorical interpretations (susceptible, intermediate, and resistant). With respect to gentamicin, no significant differences were noted among the results of disk diffusion, broth microdilution, Sensititre Auto breakpoint, or Vitek methods which characterized 60 to 67% of isolates as susceptible, 16 to 22% as intermediate, and 13 to 17% as resistant. In contrast, agar dilution, Autobac, and Avantage, although yielding gentamicin results similar to those of one another, were each significantly different in result reporting from the other four methods above for gentamicin results, and they characterized the Pseudomonas population largely as susceptible (88 to 97%), with 0 to 6% intermediate and only 3% to 6% resistant. More isolates were characterized as being resistant to gentamicin in the Avantage test if an assay broth supplemented with increased amounts of calcium was used. Cation impregnation of Autobac disks did not appreciably change Autobac results. The geometric mean MIC of gentamicin was 4 micrograms/ml lower in the agar dilution method than in the CSMHB microdilution method, despite monitoring of the agar for cation content through performance disk diffusion testing with P. aeruginosa ATCC 27853. Tobramycin activity was greater than gentamicin activity, and susceptibility to tobramycin ranged from 89 to 97%, with few statistically significant differences noted among the seven methods studied. Differences in MIC distribution and geometric mean MIC between agar dilution and CSMHB microdilution testing were minimal and suggested less of a cation influence on tobramycin than gentamicin results. Although amikacin was also more active than gentamicin (83 to 99% of isolates were susceptible), differences in the amikacin results among methods tended to reflect the same trends in reporting as seen with gentamicin testing, with the exception that results of Avantage testing were similar to those of disk diffusion, CSMHB microdilution, Sensititre, and Vitek. A difference in geometric mean MIC of 5 micrograms/ml between CSMHB testing and agar dilution testing suggested the influence of divalent cations on amikacin results. Few highly significant differences were noted among methods when isolates were tested against carbenicillin and piperacillin, except that Avantage piperacillin results (66% susceptible) and Autobac piperacillin results (98% susceptible) were noticeably different from the percent piperacillin susceptibility (range, 85 to 92%) measured by the other methods. Method-dependent variability among aminoglycoside susceptibility results, particularly when testing gentamicin, prevents meaningful comparison of Pseudomonas susceptibility trends among hospitals when different methods are used and promotes confusion and frustration among clinical microbiologists and clinicians owing to the uncertainties of clinical meaning of these data.     

Ultra-rapid flow cytometry assay for colistin MIC determination in Enterobacterales,Pseudomonas aeruginosa and Acinetobacter baumannii

ObjectivesBoth EUCAST and CLSI recommend broth microdilution for antimicrobial susceptibility testing of colistin, but this method is cumbersome and takes 16-24 h to give results. Our objective was to evaluate a rapid quantitative colistin MIC susceptibility assay based on flow cytometry analysis (FASTcolistin MIC) in comparison with standard broth microdilution assay.MethodsOne hundred and sixteen Gram-negative bacilli (78 Enterobacterales, 28 Pseudomonas aeruginosa and 10 Acinetobacter baumannii) were studied in parallel using standard broth microdilution following EUCAST recommendations and FASTcolistin MIC kit. In the last one, a bacteria suspension (0.5 MacFarland) was prepared, diluted in Muller-Hinton broth, incubated in the susceptibility panel containing different colistin concentrations (range 0.125-64 mg/L) with a fluorescent probe and incubated 1 h at 35ºC. After that, a flow cytometry analysis using CytoFLEX (Beckmam) was performed. Using a dedicated software (BioFAST) an automated MIC result was obtained after 1.5 h. Performance evaluation was performed according to the ISO standard 20776-2. Reproducibility and repeatability, categorical (CA) and essential agreement (EA), and lot-to-lot variation and operator-to-operator variability, as well as time to results were determined.ResultsOverall, 100% CA (CI 97-100%) and 95.7% EA (CI 90-98%) was obtained with high repeatability (100%; CI 80-100%)and reproducibility (97%; (CI 83-99%)). Absence of lot-to-lot variations or differences in the operators' performance was observed.ConclusionsFASTcolistin MIC is an accurate, reliable and ultra-rapid method (1 h incubation versus 24 h) for susceptibility testing of colistin of common Gram-negative bacilli recovered in clinical laboratories.     

Evaluation of in vitro methods for testing susceptibility of anaerobes to ampicillin-sulbactam and amoxicillin-clavulanic acid.

A total of 97 anaerobic bacteria were tested for susceptibility to ampicillin, ampicillin-sulbactam, and amoxicillin-clavulanic acid by broth microdilution and disk elution methods, the results of which were compared with those of the reference agar dilution method. With the broth microdilution method, approximately 95% of MICs were within 1 dilution of those of the reference agar method, with a definite (0.6 to 0.7 dilution) trend toward lower MICs. The disk elution test performed satisfactorily, but additional anaerobic isolates resistant to ampicillin-sulbactam and/or amoxicillin-clavulanic acid (currently rare) are needed to assure the predictability of resistance by the disk elution test.     

Performance characteristics of newer MIC gradient strip tests compared with the Etest for antimicrobial susceptibility testing of Neisseria gonorrhoeae

For Neisseria gonorrhoeae susceptibility testing, Etest, comparable to agar dilution, is frequently used. In recent years, newer MIC gradient strip tests have been commercialized. However, these tests have not been appropriately evaluated for gonococci. We evaluated the sensitivity, specificity, accuracy, quality, availability of antimicrobials and cost of the MIC Test Strip (Liofilchem), M.I.C.Evaluator (Oxoid) and Ezy MIC Strip (HiMedia), compared to the reference Etest (bioMérieux), for gonococcal susceptibility testing. The MICs of eight antimicrobials in 103 gonococcal international reference strains (n = 29) and clinical isolates (n = 74) were examined. Coefficient of determination (R²), complete agreement, essential agreement, SIR categorical agreement, sensitivity, specificity and accuracy were calculated. R² of the MICs for the antimicrobials ranged between 0.674–0.996, 0.617–0.993, and 0.643–0.994 for the MIC Test Strip, M.I.C.Evaluator strips and Ezy MIC Strips respectively. The essential agreement (SIR categorical agreement) was 99.6% (88.6%), 100% (87.1%) and 93.0% (83.1%) respectively. M.I.C.Evaluator strips for gonococcal key antimicrobials were lacking and the Ezy MIC Strips showed an inconsistent accuracy, quality and some strips were contaminated. The Liofilchem MIC Test Strips had limitations, but might be relatively accurate alternatives to Etest for gonococci. Strict quality assurance (at manufacturing and testing laboratory), including quality controls, are required.     

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.     

Evaluation of the E test in testing susceptibility ofPseudomonas aeruginosa to tobramycin

The E test, a new technique for measuring MICs of antimicrobial agents with the ease of disc diffusion tests, was evaluated in testing the susceptibility of 94 clinical isolates ofPseudomonas aeruginosa to tobramycin. The use of the E test was found acceptable; 93 % of the MIC results were within one log₂ dilution step and 100 % were within two log₂ dilution steps when the MICs obtained by the E test were compared to those obtained by the conventional agar dilution method. When the E test was compared to the broth microdilution method the corresponding figures were 84 % and 100 %, respectively.     

Performance of eight methods, including two new rapid methods, for detection of oxacillin resistance in a challenge set of Staphylococcus aureus organisms

Using a set of 55 Staphylococcus aureus challenge organisms, we evaluated six routine methods (broth microdilution, disk diffusion, oxacillin agar screen, MicroScan conventional panels, MicroScan rapid panels, and Vitek cards) currently used in many clinical laboratories and two new rapid methods, Velogene and the MRSA-Screen, that require less than a day to determine the susceptibility of S. aureus to oxacillin. The methods were evaluated by using the presence of the mecA gene, as detected by PCR, as the "gold standard." The strains included 19 mecA-positive heterogeneously resistant strains of expression class 1 or 2 (demonstrating oxacillin MICs of 4 to >16 microg/ml) and 36 mecA-negative strains. The oxacillin MICs of the latter strains were 0.25 to 4 microg/ml when tested by broth microdilution with 2% NaCl-supplemented cation-adjusted Mueller-Hinton broth as specified by the NCCLS. However, when tested by agar dilution with 4% salt (the conditions used in the oxacillin agar screen method), the oxacillin MICs of 16 of the mecA-negative strains increased to 4 to 8 microg/ml. On initial testing, the percentages of correct results (% sensitivity/% specificity) were as follows: broth microdilution, 100/100; Velogene, 100/100; Vitek, 95/97; oxacillin agar screen, 90/92; disk diffusion, 100/89; MicroScan rapid panels, 90/86; MRSA-Screen, 90/100; and MicroScan conventional, 74/97. The MRSA-Screen sensitivity improved to 100% if agglutination reactions were read at 15 min. Repeat testing improved the performance of some but not all of the systems.     

Mechanisms of resistance to carbapenems in meropenem- resistant Acinetobacter isolates from clinical samples

Purpose: To analyze the resistance mechanisms in Acinetobacter species by phenotypic methods. Methods: Antibiotic susceptibility profile for 150 clinical isolates of Acinetobacter was determined by the standard disk diffusion method. Isolates detected to be meropenem resistant were tested further by broth microdilution minimum inhibitory concentration (MIC) for meropenem. The resistant isolates were also tested for metallo β-lactamase (MBL) production by the double-disk approximation test, for AmpC beta-lactamase production and efflux pump detection by agar microdilution MIC with and without reserpine. Results: Twenty-one isolates were found resistant to meropenem by the standard disk diffusion method. Nine samples were from patients admitted in intensive care units (ICUs). Broth microdilution MICs of the isolates revealed low-level resistance to meropenem. MBL was not produced by any of these isolates. AmpC β-lactamases were produced by nine (43%) isolates. ‘Efflux pump’-mediated resistance to meropenem was detected in two out of nine random isolates tested for the same. Conclusions: Carbapenem resistance is not uncommon in Acinetobacter isolates. AmpC production may cause carbapenem resistance. MBL and efflux pump may not be important causes of carbapenem resistance.     

Area of technical uncertainty for susceptibility testing of amoxicillin/clavulanate against Escherichia coli: analysis of automated system,Etest and disk diffusion methods compared to the broth microdilution reference

ObjectivesThe European Committee on Antimicrobial Susceptibility Testing (EUCAST) recently warned about an area of technical uncertainty (ATU) of amoxicillin/clavulanate (AMX/C) disk susceptibility testing against members of the Enterobacterales. Thus, we aimed to compare the reliability of three routine methods and to evaluate the impact of the ATU.Methods286 Escherichia coli strains (including 159 AMX-resistant strains) were categorized for the two EUCAST AMX/C breakpoints by disk diffusion (Bio-Rad), the Phoenix automated system (Becton Dickinson) and the Etest (AES) compared to the broth microdilution reference method.ResultsBy microdilution, 84.2% of strains were AMX/C-susceptible using the urinary breakpoint (MIC ≤32 mg/L) and 62.2% using the systemic breakpoint (MIC ≤8 mg/L), with 63.6% of MICs between 4 and 16 mg/L. For the systemic breakpoint, category agreement (CA) and very major error (VME) were unacceptable for the Etest (71.7% and 27.3%), disk (73.1% and 23.4% at 19-mm cut-off) and to a lesser extent for the Phoenix system (83.6% and 10.5%). For disks, an unacceptable VME rate was observed for diameters up to 22 mm, probably due to overcharged disks. For the Etest, VMEs were high at 6 mg/L (46/63) and 8 mg/L (22/29). For the urinary breakpoint, CA was more acceptable for disk (88.9%) and Etest (84.3%) but was unevaluable for Phoenix.ConclusionAMX/C susceptibility testing of E. coli for systemic breakpoint was unreliable with the three routine methods, explained mainly by the high prevalence (~60%) of strains with microdilution MICs around the breakpoint (8 mg/L). Our data confirmed the EUCAST 19–20-mm ATU for disk and suggest introducing ATU for Etest MIC values of 6 and 8 mg/L.     

Interest of the disk diffusion method for screening Clostridium difficile isolates with decreased susceptibility to antibiotics

AimIn vitro determination of Clostridium difficile susceptibility to antibiotics is not routinely performed. The aim of this study was to evaluate the performance of antibiotic susceptibility determination with the disk diffusion method for screening C. difficile isolates with decreased susceptibility to antibiotics.MethodsThirty-six C. difficile isolates (toxigenic or not) isolated in 2005 and 2006 from three hospitals Assistance publique–Hôpitaux de Paris (Jean-Verdier, René-Muret, Beaujon) were studied by disk diffusion method with 14 antibiotics. Mueller-Hinton agar supplemented with sheep blood (Bio-Rad*) were swabbed with a C. difficile suspension at 1 McFarland. To check the results obtained with the disk diffusion method, Minimal Inhibitory Concentration (MIC) were performed respectively with E-test for glycopeptides and metronidazole and with the agar dilution reference method and E-test for new molecules with a potential activity against anaerobes: imipenem, ertapenem, linezolid and moxifloxacin.ResultsThe decreased susceptibility (resistant and intermediate) observed was 40% for amoxicillin–clavulanate, 60% for piperacillin–tazobactam, 100% for ceftriaxone, 81% for imipenem, 61% for ertapenem, 2% for chloramphenicol, 34% for erythromycin, 90% for lincomycin, 2% for linezolid, 98% for levofloxacin, 17% for moxifloxacin and 0% for vancomycin, teicoplanin and metronidazole. The results obtained with the disk diffusion method were compared to MICs obtained with E-test and reference method.ConclusionThe disk diffusion method seems to be a good method to detect isolates suspected to have a decreased susceptibility and consequently to reduce MIC determinations.     

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.