Dilution Reference Method Research Articles

Wild-type MIC distributions and epidemiological cutoff values for 5-flucytosine and Candida species as determined by EUCAST broth microdilution.

EUCAST has established clinical breakpoints and epidemiological cutoff values (ECOFFs) for Candida spp. However, limited data are available for 5-flucytosine (5-FC). We assessed the in vitro susceptibility of 5-FC against a large collection of clinical Candida species using EUCAST methodology and determined the associated ECOFFs. A total of 5622 Candida isolates were collected from patients across the Netherlands between 2008 and 2024. 5-FC MICs were determined using the EUCAST microbroth dilution reference method. Furthermore, MICs were extracted from the EUCAST website. The MICs from this study and those extracted were used to determine ECOFFs and local ECOFFs (L-ECOFFs). 5-FC exhibited potent in vitro activity against C. albicans, N. glabratus and C. parapsilosis, while decreased susceptibility was observed for C. tropicalis, Pichia species, K. marxianus, Y. lipolytica, and C. auris. The ECOFFs (mg/L) and the percentages of WT isolates for 5-FC were: C. albicans: 0.5 (97.2%), N. glabratus: 0.5 (96.6%), C. parapsilosis: 0.5 (99.5%) and P. kudriavzevii: 8 (99.4%). The L-ECOFF (mg/L) and the percentages of WT isolates for 5-FC were: C. dubliniensis: 0.25 (96.8%), C. tropicalis: 0.25 (67.2%), K. marxianus: 0.25 (48.0%), C. lusitaniae: 0.25 (86.5%), M. guillermondii: 0.125 (95.9%) and P. norvegiensis: 8 (94.2%). 5-FC remains a valuable drug to manage difficult-to-treat invasive Candida infections. In vitro susceptibility cannot be predicted based on species identification for most Candida species, but requires MIC-testing. ECOFFs will help to interpret the MICs to support treatment decisions.

Evaluation of several routine methods for fosfomycin and mecillinam susceptibility testing of Enterobacterales urine isolates.

Performance evaluation of routine laboratory methods to determine the susceptibility of Enterobacterales urinary isolates to fosfomycin (oral administration) and mecillinam. We collected 347 Enterobacterales isolates from monomicrobial midstream urine samples from women with significant bacteriuria and leukocyturia. Mostly non-Escherichia coli isolates (i.e. Klebsiella spp., Citrobacter koseri, Enterobacter cloacae complex and Proteus mirabilis) were included (n = 298). Performance of VITEK®2, ETEST®, and disc diffusion to determine fosfomycin and mecillinam susceptibility was evaluated following International Organization for Standardization (ISO) 20776-2:2021 (or 20776-2:2007 for disc diffusion) in comparison with the agar dilution reference method. For fosfomycin testing, VITEK®2 and ETEST® were close to reaching ISO requirements (essential agreement ≥ 90%; bias ±30%) for C. koseri, E. coli and P. mirabilis. Categorical agreement (CA) and major error rates were acceptable for disc diffusion. Fosfomycin displayed lower activity against E. cloacae complex and Klebsiella spp., with MIC50 (minimum inhibitory concentration required to inhibit the growth of 50% of tested isolates) equal to the E. coli EUCAST breakpoint (8 mg/L). For these species, the three alternative techniques overestimated MICs and resistance, and did not meet performance criteria. For mecillinam testing of Enterobacterales isolates, apart from P. mirabilis, ETEST® nearly fulfilled ISO requirements, and CA rates were acceptable for disc diffusion. ISO criteria were reached for C. koseri and E. coli testing with VITEK®2, apart from too high rates of very major errors. For P. mirabilis, performances were unacceptable, whatever the routine method used. Commercially available tests may serve as alternatives to agar dilution to assess fosfomycin (oral) and mecillinam susceptibility of Enterobacterales urinary isolates, with important interspecies variabilities. Additional studies comprising more fosfomycin- and mecillinam-resistant isolates are needed to strengthen our conclusions.

Skipped wells and scientific error during fosfomycin agar dilution and broth microdilution lead to inconsistent minimal inhibitory concentrations and may be cause for reevaluating testing methods for Klebsiella pneumoniae.

Despite the first-line recommendation of fosfomycin for uncomplicated urinary tract infections (UTIs), there are pressing barriers for optimizing its use for the treatment of non-Escherichia coli Enterobacterales UTI. There are no approved breakpoints for oral use against other Enterobacterales, and the recommended agar dilution (AD) reference method for minimal inhibitory concentration (MIC) determination is largely impractical. Using 160 clinical Klebsiella pneumoniae isolates, we sought to understand rates of skipped wells and MIC imprecision in broth microdilution (BMD) and how that compares to rates of error using AD. Though the Clinical and Laboratory Standards Institute refers to the skipped well phenomena in their recommendation against the use of BMD, there is a paucity of data on its frequency. While AD and BMD produced similar MIC50/90 values (32/256 µg/mL for AD and 64/256 µg/mL for BMD), essential agreement was poor. No-growth wells at concentrations below the MIC occurred in up to 10.9% of wells at a given concentration, as the most frequent scientific error. Growth in concentrations above the measured MIC occurred in up to 3.3% of wells and was seen within three dilutions of the MIC for BMD. Observation of single colonies either at or beyond the measured MIC for AD was also common and occurred up to 8.3% and 2.5% of the time, respectively. The frequent scientific error in both testing methods should prompt re-evaluation of AD guidelines and expansion of MIC testing methods for fosfomycin susceptibility testing, as poor agreement with another method prone to scientific error should not be the main detractor from BMD use.IMPORTANCEDespite the recommendation of fosfomycin for uncomplicated urinary tract infections (UTIs), there are barriers for optimizing its use. There are no approved breakpoints for oral use against other Enterobacterales, and the recommended agar dilution (AD) reference method for MIC determination is largely impractical. The use of broth microdilution (BMD) for fosfomycin testing is not recommended by the Clinical and Laboratory Standards Institute due to unsatisfactory precision and skipped wells-occurrence of no-growth in a single well before the minimal inhibitory concentration (MIC)-and trailing endpoints. We sought to understand rates of skipped wells and growth at concentrations above measured MICs in BMD and how that compares to scientific error using AD. No-growth wells at concentrations below the MIC occurred in up to 10.9% of wells for BMD and single colonies at or beyond measured MICs for AD were also common. Frequent scientific error in both methods should prompt re-evaluation of both AD and BMD for fosfomycin susceptibility testing.

Assessment of the gradient diffusion method for fosfomycin susceptibility testing in Staphylococcus spp. and Enterococcus spp. isolated from the urine of companion dogs in Thailand.

The agar dilution method is the approved method for determining the minimum inhibitory concentration (MIC) in fosfomycin susceptibility testing, whereas the broth dilution method is not recommended. This study aimed to investigate the potential of the gradient diffusion method as a more convenient alternative to agar dilution method for MIC evaluation, particularly for the susceptibility testing of Staphylococcus spp. and Enterococcus spp. to fosfomycin. A total of 194 isolates of Staphylococcus spp. and Enterococcus spp. were collected from urine samples of dogs diagnosed with bacterial cystitis. Bacterial identification and susceptibility to multiple antibiotics were tested using the Vitek 2 automated system. The susceptibility to fosfomycin was compared between agar dilution (reference method) and the gradient diffusion method. We assessed the agreement rates and errors between the two approaches by analyzing the MIC data. Staphylococcus pseudintermedius (98.7%) and Enterococcus faecalis (80.0%) exhibited high fosfomycin susceptibility rates, whereas Enterococcus faecium exhibited a lower susceptibility rate (38.5%). The gradient diffusion method demonstrated unacceptably low essential agreement (EA) rates (>90%) but acceptable categorical agreement (CA) rates (≥ 90%) for S. pseudintermedius (83.54% EA and 97.47% CA) and coagulase-negative staphylococci (CoNS) such as Staphylococcus chromogenes, Staphylococcus hominis, and Staphylococcus simulans (85.00% EA and 95.00% CA). Enterococcus spp. had an acceptable EA of 93.75%, but an unacceptably low CA rate of 82.81%, with a minor error rate of 17.19%. No significant errors were observed for Staphylococcus and Enterococcus spp. The gradient diffusion method reliably determines MICs and interpretative breakpoints (S, I, R) for S. pseudintermedius. However, its applicability to CoNS and enterococci may be limited due to unacceptable errors.

Fungal keratitis in Iran: Risk factors, clinical features, and mycological profile.

This study was intended to investigate the clinical features and predisposing factors of fungal keratitis (FK), as well as molecular identification and antifungal susceptibility of causative agents in Tehran, Iran. This cross-sectional study was carried out from April 2019 to May 2021. All fungi isolates were identified using conventional methods and were confirmed by DNA-PCR-based molecular assays. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) was used to identify yeast species. Minimum inhibitory concentrations (MIC) of eight antifungal agents were assessed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) microbroth dilution reference method. Fungal etiology was confirmed in 86 (7.23%) of 1189 corneal ulcers. A significant predisposing factor for FK was ocular trauma caused by plant materials. Therapeutic penetrating keratoplasty (PKP) was required in 60.4% of cases. The predominant fungal species isolated was Fusarium spp. (39.5%) followed by Aspergillus spp. (32.5%) and Candida spp. (16.2%). The MIC results indicate that amphotericin B may be appropriate for treating FK caused by Fusarium species. FK caused by Candida spp. can be treated with flucytosine, voriconazole, posaconazole, miconazole, and caspofungin. In developing countries such as Iran, corneal infection due to filamentous fungi is a common cause of corneal damage. In this region, fungal keratitis is observed primarily within the context of agricultural activity and subsequent ocular trauma. Fungal keratitis can be managed better with understanding the "local" etiologies and antifungal susceptibility patterns.

Broth Microdilution and Gradient Diffusion Strips vs. Reference Agar Dilution Method: First Evaluation for Clostridiales Species Antimicrobial Susceptibility Testing.

Antimicrobial susceptibility testing of anaerobes is challenging. Because MIC determination is recommended by both CLSI and EUCAST, commercial broth microdilution and diffusion strip tests have been developed. The reliability of broth microdilution methods has not been assessed yet using the agar dilution reference method. In this work, we evaluated two broth microdilution kits (MICRONAUT-S Anaerobes® MIC and Sensititre Anaerobe MIC®) and one gradient diffusion strip method (Liofilchem®) for antimicrobial susceptibility testing of 47 Clostridiales isolates (Clostridium, Clostridioides and Hungatella species) using the agar dilution method as a reference. The evaluation focused on comparing six antimicrobial molecules available in both microdilution kits. Analytical performances were evaluated according to the Food and Drug Administration (FDA) recommendations. Essential agreements (EA) and categorical agreements (CA) varied greatly according to the molecule and the evaluated method. Vancomycin had values of essential and categorical agreements above 90% for the three methods. The CA fulfilled the FDA criteria for three major molecules in the treatment of Gram-positive anaerobic infections (metronidazole, piperacillin/tazobactam and vancomycin). The highest rate of error was observed for clindamycin. Multicenter studies are needed to further validate these results.

Triazole-Resistance in Environmental Aspergillus fumigatus in Latin American and African Countries.

Triazole-resistance has been reported increasingly in Aspergillus fumigatus. An international expert team proposed to avoid triazole monotherapy for the initial treatment of invasive aspergillosis in regions with >10% environmental-resistance, but this prevalence is largely unknown for most American and African countries. Here, we screened 584 environmental samples (soil) from urban and rural locations in Mexico, Paraguay, and Peru in Latin America and Benin and Nigeria in Africa for triazole-resistant A. fumigatus. Samples were screened using triazole-containing agars and confirmed as triazole-resistant by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth dilution reference method. Isolates were further characterized by cyp51A sequencing and short-tandem repeat typing. Fungicide presence in samples was likewise determined. Among A. fumigatus positive samples, triazole-resistance was detected in 6.9% (7/102) of samples in Mexico, 8.3% (3/36) in Paraguay, 9.8% (6/61) in Peru, 2.2% (1/46) in Nigeria, and none in Benin. Cyp51A gene mutations were present in most of the triazole-resistant isolates (88%; 15/17). The environmentally-associated mutations TR34/L98H and TR46/Y121F/T289A were prevalent in Mexico and Peru, and isolates harboring these mutations were closely related. For the first time, triazole-resistant A. fumigatus was found in environmental samples in Mexico, Paraguay, Peru, and Nigeria with a prevalence of 7–10% in the Latin American countries. Our findings emphasize the need to establish triazole-resistance surveillance programs in these countries.

Susceptibility of Clinical Enterobacterales Isolates With Common and Rare Carbapenemases to Mecillinam.

Purpose: To investigate the susceptibility of carbapenemase-producing Enterobacterales (CPE) to mecillinam based on the recently updated European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for uncomplicated Urinary Tract Infection (uUTI).Methods: The challenge collection consisted of 105 molecularly characterized Enterobacterales [Klebsiella spp. (N = 49), Escherichia coli (N = 30), Enterobacter cloacae (n = 13), Citrobacter freundii (N = 9), Proteus mirabilis (N = 3), and Raoultella ornithinolytica (N = 1)]. Isolates produced OXA-48 (N = 18), OXA-48-like (N = 18), VIM (N = 22), NDM (N = 22), KPC (N = 12), IMI (N = 9), IMP (N = 6), GES (N = 1), OXA-58 (N = 2) or combinations thereof (N = 5). MICs of carbapenems were determined by agar gradient diffusion (AGD). MICs of mecillinam were assessed by agar dilution (reference method) and compared to disk diffusion (DD) and AGD.Results: Overall 23/105 CPE (21.9%) were susceptible to mecillinam. Susceptibility was observed in E. coli (N = 12), E. cloacae (N = 7), and Klebsiella pneumoniae (N = 4) producing IMI, OXA-48, OXA-48-like, and NDM-1 carbapenemases. MIC50 for mecillinam in all isolates was 128 mg/L while MIC50 for meropenem was 8 mg/L. Lower MICs for mecillinam were found in IMI (MIC50 8 mg/L) and OXA-48-like (MIC50 16 mg/L) producers. The comparison of the different susceptibility methods showed very major errors of 12.2% with AGD and 8.5% with disk diffusion when compared to the reference method.Conclusion: Mecillinam susceptibility was restricted to isolates producing IMI-, OXA-48-like, and NDM-1 carbapenemases and was documented despite high carbapenem MICs in some isolates. Mecillinam could be a promising oral antimicrobial in uUTI caused by E. coli and E. cloacae isolates carrying IMI- and OXA-48-like carbapenemases; however, susceptibility testing by AGD and disk diffusion remains problematic.

1222. Fosfomycin Susceptibility Testing using the new ETEST®FO

BackgroundFosfomycin (FO) is a bactericidal antibiotic with a broad spectrum of activity against a wide range of Gram-positive and Gram-negative bacteria. Oral FO is mainly used in the treatment of urinary tract infections, particularly those caused by E. coli and E. faecalis. In order to determine MICs to FO, the ETEST® FM is already available but the reading can be difficult especially with E. coli. To resolve this issue, a new ETEST® with FO, called ETEST® FO, has been developed (not FDA cleared, yet). The purpose of this study is to compare this new strip to the agar dilution reference method (AD) on a panel of E. coli and E. faecalis.MethodsA total of 39 isolates comprising 20 E. coli (ESBL or CPE) and 19 E. faecalis (VRE or VSE) were tested by ETEST® FO and Agar dilution. The isolates were sub-cultured on Columbia agar plates supplemented with 5% sheep blood before testing. After incubation, suspensions of the isolates were prepared in 0.85% saline. These suspensions were used to inoculate both AD and ETEST® plates. Results were read after 16-20 hours incubation at 35°C +2°C in ambient air. Following CLSI QC guideline, 4 QC organisms were tested. Results were analyzed using the FDA/CLSI breakpoints for FO (S < 64µg/mL, I=128 µg/mL, R> 256 µg/mL). Performance was evaluated using FDA performance criteria, essential agreement (EA, ≥ 90%), category agreement (CA, ≥ 90%), major error rate (ME, ≤3.0%) and very major error rate (VME, ≤2.0%).ResultsAll the QC strains MICs were within the CLSI ranges. For the panel results, see the table below:Performance for ETEST® FO on E. coli and E. faecalisSpeciesEACAVery Major Error RateMajor Error RateMinor Error RateOverall (39)97.4% (38/39)97.4% (38/39)0% (0/2)0.0% (0/37)2.6 % (1/39) E. faecalis (19)100% (19/19)94.7% (18/19)NA (No R)0.0% (0/19)5.2% (1/19) E. coli (20)95.0% (19/20)100% (20/20)0% (0/2)0.0% (0/18)0% (0/20)ConclusionThis first and preliminary study shows that ETEST® FO can potentially meet the FDA acceptance criteria and could be a valuable tool for determining FO MIC for E. coli harboring various resistance mechanisms and E. faecalis including VRE. Moreover, in comparison with the current ETEST FM strip, this new strip brings a real reading improvement and resolve the issue for E. coli. The clinical study phase will determine the product’s performance.DisclosuresMarion Pompilio, BioMérieux (Employee) Gilles Zambardi, biomerieux (Employee)

In vitro fosfomycin study on concordance of susceptibility testing methods against ESBL and carbapenem-resistant Enterobacteriaceae

ObjectivesThe increasing emergence and diffusion of multidrug-resistant (MDR) pathogenic bacteria, both in hospital and community settings, is inducing clinicians to reconsider old antibiotics, such as fosfomycin, to overcome the difficulties posed by these microorganisms. Recent studies have reported good in vitro activity of fosfomycin against extended spectrum ß-lactamases (ESBL) and carbapenem-resistant Enterobacteriaceae. The aim of this study was to assess thein vitro activity of fosfomycin by different methods against 120 clinical MDR isolates. MethodsFosfomycin minimum inhibitory concentrations were determined using the agar dilution reference method (AD), gradient test (GT), broth microdilution method (BMD), according to CLSI recommendations, and automated systems (VITEK 2 and BD Phoenix) against 85 carbapenem-resistant Klebsiella pneumoniae and 35 ESBL-producing Escherichia coli. Agreement and discrepancies between the evaluated methods and the reference method were calculated. ResultsFosfomycin showed very good activity against ESBL-producing E. coli (88.6%). Excellent agreement (100%) between the three (AD, BMD and GT) susceptibility methods was found for E. coli. No major errors were observed. The fosfomycin resistance rate ranged from 24% (KPC-producing) to 100% (NDM-OXA-48 co-producing) K. pneumoniae. For all carbapenem-resistant K. pneumoniae strains, categorical agreement was >90% for all methods except for VITEK 2, which was 84%. ConclusionsWhen ESBL E. coli isolates are found to be susceptible to fosfomycin with automated systems, it is not necessary to verify these results with the AD reference method; while for resistant strains, the GT can be used. In cases of KPC K. pneumoniae resistant to fosfomycin, the AD method is the only reference method.

Susceptibility of Clinical Isolates of Escherichia coli to Fosfomycin as Measured by Four In Vitro Testing Methods.

Clinical isolates of Escherichia coli (n = 554) were tested against fosfomycin using agar dilution, disk diffusion, and Etest. Agar dilution (reference method) identified few isolates with fosfomycin MICs of 64 (n = 3), 128 (n = 4), and ≥256 μg/ml (n = 2). Applying CLSI (M100, 2020) and EUCAST (v. 10.0, 2020) breakpoints, 98.9% and 98.4% (agar dilution), 99.3% and 99.1% (disk diffusion), and 99.1% and 98.9% (Etest) of isolates were fosfomycin susceptible, respectively. Essential agreement (agar dilution versus Etest) was low (40.8%); 59.3% (131/221) of isolates with agar dilution MICs of 2 to 128 μg/ml tested 2 to 4 doubling dilutions lower by Etest. Applying CLSI breakpoints, categorical agreement was >99% for both disk diffusion and Etest; no major errors (MEs) or very major errors (VMEs) were identified, and rates of minor errors (mEs) were <1%. EUCAST breakpoints yielded categorical agreements of >99% and no MEs for both disk diffusion and Etest; however, VMEs occurred at unacceptable rates of 44.4% (disk diffusion) and 33.3% (Etest). All isolates with agar dilution MICs of ≥32 μg/ml (n = 12) and a subset of isolates with MICs of ≤16 μg/ml (n = 49) were also tested using the Vitek 2 AST-N391 card and generated fosfomycin MICs 1 to ≥3 doubling dilutions lower than agar dilution for 11/12 isolates with agar dilution MICs of ≥32 μg/ml. We conclude that performing fosfomycin disk diffusion or Etest on urinary isolates of E. coli and interpreting results using CLSI breakpoints reliably identified fosfomycin-susceptible isolates regardless of differences in endpoint reading criteria. EUCAST breakpoints generated excessive rates of VMEs for our isolate collection of high fosfomycin susceptibility.

Beta-lactams susceptibility testing of penicillin-resistant, ampicillin-susceptible Enterococcus faecalis isolates: a comparative assessment of Etest and disk diffusion methods against broth dilution

This study aimed to evaluate the accuracy of disk diffusion and Etest methods, compared to that of the broth dilution reference method for identifying beta-lactam susceptibilities of Penicillin-Resistant, Ampicillin-Susceptible Enterococcus faecalis (PRASEF) isolates. Fifty-nine PRASEF and 15 Penicillin-Susceptible, Ampicillin-Susceptible E. faecalis (PSASEF) clinical nonrepetitive isolates were evaluated. The effectiveness of five beta-lactams (ampicillin, amoxicillin, imipenem, penicillin, and piperacillin) was tested. All antimicrobial susceptibility tests were performed and interpreted according to the Clinical and Laboratory Standards Institute guidelines. Interpretative discrepancies, such as essential agreement, categorical agreement, and errors, were assessed. The acceptability was ≥ 90% for both categorical agreement and essential agreement. Etest proved to be an accurate method for testing beta-lactam susceptibilities of the emerging PRASEF isolates, disk diffusion presented poor performance, particularly for imipenem and piperacillin.

Comparison of minimum inhibitory concentration results for gepotidacin obtained using agar dilution and broth microdilution methods

Gepotidacin (GSK2140944) is a first in class, novel triazaacenaphthylene bacterial type II topoisomerase inhibitor in Phase 3 clinical development for the treatment of gonorrhea and uncomplicated urinary tract infections (acute cystitis). This study tested the equivalency of minimal inhibitory concentrations (MICs) obtained by two reference susceptibility testing methods, agar dilution and broth microdilution, for gepotidacin when tested against various gram-positive and gram-negative organisms. Equivalency, measured as the essential agreement >89.9%, was established between the two methods for determining gepotidacin susceptibility results against Staphylococcus spp., Streptococcus spp., and Escherichia coli. However, for Neisseria gonorrhoeae and Haemophilus influenzae, equivalency was not established. Agar dilution remains the sole dilution reference method for determining gepotidacin MICs against N. gonorrhoeae.

2166. Performance Study on the New ETEST® Piperacillin/Tazobactam (P/T) MIC Strip

BackgroundPiperacillin/Tazobactam combination is a first-line antibiotic and carbapenem sparing option for severe infections due to Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. ETEST® strips allow to determine antimicrobial Minimum Inhibitory Concentration (MIC). ETEST® Piperacillin/Tazobactam (Ptc) was developped in 1995 against agar dilution reference method. Since then, resistance to Piperacillin/Tazobactam has been increasing and broth microdilution (BMD) substituted for agar dilution as the reference method. The new ETEST® P/T strip for determining MIC of Enterobacteriaceae, P. aeruginosa and A. baumannii was developed against BMD using a panel of recent strains well genotypically characterized. The aim of this study was to compare the performance of both strips on a panel of challenging strains harboring different-resistant mechanisms.MethodsA total of 64 strains were tested using ETEST® P/T, ETEST® PTc and BMD: 48 Enterobacteriaceae including 25 resistant strains and 16 P. aeruginosa including 11 resistant strains. The results were analyzed for essential (EA) and category (CA) agreements, minor (mE), major (ME) and very major (VME) error rates using FDA/CLSI 2019 breakpoints (Enterobacteriaceae, P. aeruginosa: ≤ 16/4(S); ≥ 128/4(R) µg/mL).ResultsAlthough the panel of strains was challenging including different resistant mechanisms (acquired penicillinase, high-level cephalosporinase, acquired cephalosporinase, ESBL, carbapenemase), the new ETEST® P/T performance was significantly improved for Enterobacteriaceae with an EA at 92,2% without ME or VME. This improvement was also linked to the easiest reading (significant decrease of microcolonies in the ellipse zone). For P. aeruginosa, the performance was similar between the two strips but the new ETEST® P/T was better correlated with the BMD and showed an EA of 100%. The results are summarized in the table.ConclusionThe new ETEST® P/T improved the MIC determination and resistance detection, as well as the reading of MIC end points for the routine use. This study emphasizes the need to check the performance of the antimicrobial susceptibility testing products by testing strains reflecting the current epidemiology. Disclosures All authors: No reported disclosures.

Accuracy and reproducibility of the Etest to detect drug-resistant Neisseria gonorrhoeae to contemporary treatment.

Neisseria gonorrhoeae is a sexually transmitted bacterial pathogen that continues to evolve to become resistant to known antibiotics. In preparing for potential emergence, the Centers for Disease Control and Prevention recommends that clinical laboratories maintain or develop protocols to assess antibiotic susceptibly for this organism. This study examines the intra-laboratory variability of using the Etest method to provide consistent MIC values for N. gonorrhoeae and also compared the results of the Etest to known agar dilution MIC values. Clinical N. gonorrhoeae isolates, 100 paired duplicates, were tested by eight laboratories for antibiotic susceptibility to ceftriaxone, cefixime and azithromycin using Etest strips.Results/Key findings. Overall, >80 % of the paired Etest MIC values were within one log2 dilution of the replicate. When compared to the agar dilution reference method, the cefixime Etest MIC values were consistently underreported by one dilution (seven laboratories) or two dilutions (one laboratory). The azithromycin Etest MIC values agreed 90.7 % with the agar dilution MIC values while the agreement with ceftriaxone was 90.9 %. Overall, the Etest method yielded reproducible MIC values within each laboratory with the azithromycin and ceftriaxone MIC results consistent to the reference agar dilution method while the cefixime result tended to provide a lower MIC value.

Rifaximin-resistant Clostridium difficile strains isolated from symptomatic patients

BackgroundRifaximin has been proposed as an alternative treatment for specific cases of Clostridium difficile infection (CDI) and intestinal decontamination. Rifaximin-resistant C. difficile has occasionally been reported. Antibiotic susceptibility testing relies on anaerobic agar dilution (reference method), which is cumbersome and not routinely used. There is no commercial test for detection of resistance to rifaximin. ObjectivesTo assess resistance to rifaximin by C. difficile and to evaluate the correlation between the results of the rifampicin E-test and susceptibility to rifaximin. MethodsWe compared the in vitro susceptibility of clinical CDI isolates to rifaximin over a 6-month period using the agar dilution method with susceptibility to rifampicin using the E-test. All isolates were characterized using PCR-ribotyping. Clinical data were recorded prospectively. ResultsWe recovered 276 consecutive C. difficile isolates and found that 32.2% of episodes were caused by rifaximin-resistant strains. The MICs for rifaximin ranged from <0.0009–256 mg/L, with a geometric mean (GM) of 0.256 mg/L, an MIC50/90 of 0.015/>256 mg/L. Rifaximin and rifampicin MICs were comparable, and all strains classed as resistant by agar dilution were correctly classified as resistant by E-test. The most common ribotypes were 001 (37.2%), 078/126 (14.3%), and 014 (12.0%). Ribotype 001 exhibited the highest MICs for rifaximin. ConclusionsResistance to rifaximin was common; resistance rates were higher in ribotype 001 strains. Susceptibility to rifaximin determined by agar dilution correlated with susceptibility to rifampicin determined using the E-test, including rifaximin-resistant strains. Our results suggest that the rifampicin E-test is a valid method for the prediction of rifaximin-resistant C. difficile.

Evaluation of direct Etest for antimicrobial susceptibility testing of bacteria isolated from synovial fluid of horses using enrichment bottles

This study evaluated the Etest for direct antimicrobial susceptibility testing (AST) of bacteria from equine synovial specimens, incubated in BACTEC enrichment bottles. Ninety-four culture-positive broths were inoculated onto agar to directly determine the minimum inhibitory concentrations (MICs) of 13 antimicrobials, using the Etest (direct Etest). Results were compared with those obtained with the agar dilution reference method, the standard Etest, and the disc diffusion method, after subculture and standardisation of the inoculum. For categorical comparison of AST results, MICs were translated into susceptibility categories, using clinical breakpoints.The direct Etest predicted categorical susceptibility/resistance of bacteria from equine synovial fluid with acceptable accuracy (overall categorical agreement, 91%) and was more reliable than the disc diffusion test. The direct Etest was less accurate than the standard Etest for generating MICs ± 1 log dilution relative to the reference method (overall essential agreement, 69% vs. 89%). As the Etest generated a high percentage of inaccuracies with trimethoprim and sulfadiazine, these were less suitable antimicrobial agents for inclusion. In conclusion, the direct Etest reliably predicted the susceptibility of isolates from equine synovial fluid for the tested antimicrobials, except for trimethoprim and sulfadiazine. Since it did not require subculture and preparation of a standardised inoculum, direct Etest results were available at least 24 h earlier than with other methods, which could facilitate the diagnosis of synovial infections. However, when accuracy is prioritised over speed for MIC determination, the standard Etest is preferred over the direct Etest.

Is a New Standard Needed for Diffusion Methods for In Vitro Susceptibility Testing of Fosfomycin against Pseudomonas aeruginosa?

We analyzed fosfomycin susceptibility results in Pseudomonas aeruginosa clinical isolates obtained by MIC gradient strips and disk diffusion methods using two different inocula, 10(8) and 10(6) CFU/ml, and compared them to the agar dilution reference method. Essential and categorical agreements were 93.6% and 95%, respectively, for the 10(6) CFU/ml alternative inoculum, and they were 67.6% and 78.2%, respectively, for the standard inoculum (10(8) CFU/ml). The use of the 10(6) CFU/ml inoculum improves the agreement values and inhibition zone readings.

Comparison of the Vitek 2, MicroScan, and Etest Methods with the Agar Dilution Method in Assessing Colistin Susceptibility of Bloodstream Isolates of Acinetobacter Species from a Korean University Hospital

We evaluated three commercial colistin susceptibility testing methods using 213 bloodstream Acinetobacter isolates identified by gene sequencing. Compared to the agar dilution reference method, excellent categorical agreements (both 99.1%) were observed using Vitek 2 and Etest, compared to 87.3% (95.7% for Acinetobacter baumannii and 80.7% for non-baumannii Acinetobacter isolates) using MicroScan.

Updating Antimicrobial Susceptibility Testing Methods

1. Cynthia B. Schofield, MPH, MT (CAMT)[⇑][1] 1. VA San Diego Healthcare System (retired), San Diego, CA 1. Address for Correspondence: Cynthia B. Schofield, MPH, MT (CAMT), VA San Diego Healthcare System (retired), 7050 Weller St., San Diego, CA, (858) 450-9651, cschofield{at}san.rr.com 1. Explain the physician's choice of antibiotic agents according to CLSI guidelines. 2. Define and discuss MIC breakpoints. 3. Compare qualitative versus quantitative antimicrobial susceptibility testing (AST) methods. 4. Contrast the various quantitative AST methods. 5. Discuss the broth dilution reference method. 6. Discuss genotypic methods and their purposes. The importance of antimicrobial susceptibility testing (AST) as a clinical laboratory function escalates as organism resistance to the available antimicrobial agents increases. As patient outcome is based on antimicrobial therapy, standardization of the AST methods determining therapy is essential. The Clinical and Laboratory Standards Institute (CLSI) in the US and the European Committee on Antimicrobial Testing (EUCAST) in Europe determine these standards that are updated annually. Of concern to physicians are questions regarding the establishment of breakpoints, which are the interpretive cut-off values of minimal inhibitory concentration (MIC) in quantitative testing and inhibition zones for antimicrobial agents in qualitative testing based on geographical surveys of clinical isolates. The CLSI performance standards in the US and the EUCAST in Europe, the groups responsible for developing the breakpoints, may differ in perception and usage.1,2 Interpretation of qualitative results as susceptible (S), intermediate (I) or resistant (R) and quantitative measures (MICs) of antimicrobial activity are standardized by CLSI in the U.S. with breakpoint guidelines updated annually for each antibiotic tested.1,2,4 Antimicrobial Agents The clinical laboratory's list of antimicrobial agents is chosen by the infectious disease specialists and pharmacologists at the individual institution according to the institution's unique guidelines. A formulary of agents that physicians regularly prescribe is derived from the susceptibility testing of organisms typically isolated at each clinical laboratory and is guided by a monthly updated antibiogram. The number of agents tested is designated by the AST method used, e.g. 12 disks per 150 mm Mueller-Hinton agar plate or similar number per… ABBREVIATIONS: AST-Antimicrobial Susceptibility Testing; CLSI-Clinical and Laboratory Standards Institute; EUCAST-European Committee on Antimicrobial Susceptibility Testing; FDA-Food and Drug Administration; MIC-minimal inhibitory concentration; MH-Mueller-Hinton; CAMHB-cation-adjusted Mueller Hinton broth; ATCC-American Type Culture Collection; MRSA-methicillin-resistant Staphylococcus aureus ; VRE-vancomycin-resistant Enterococcus ; S, I, R-sensitive, intermediate, resistant; PCR-polymerase chain reaction; DNA-deoxyribonucleic acid; RT-reverse transcriptase; CAI-community-associated infection; HAI-hospital-associated infection; MSSA-methicillin-sensitive Staphylococcus sp.; PBP-penicillin-binding proteins; ESBL-extended-spectrum beta lactamase; KPC- Klebsiella pneumoniae , carbapenem-resistant; CFU-colony forming units; TSB-trypticase soy broth; CSF-cerebrospinal fluid; ESBL-extended-spectrum beta lactamase 1. Explain the physician's choice of antibiotic agents according to CLSI guidelines. 2. Define and discuss MIC breakpoints. 3. Compare qualitative versus quantitative antimicrobial susceptibility testing (AST) methods. 4. Contrast the various quantitative AST methods. 5. Discuss the broth dilution reference method. 6. Discuss genotypic methods and their purposes. [1]: #corresp-1