Search

Background: Sepsis is a life-threatening condition that impacts 49 million people annually and causes 11 million deaths worldwide. Surviving bloodstream infections (BSIs) depends on the rapid administration of effective antimicrobial treatment, underscoring a need for rapid antimicrobial susceptibility testing (AST)., Aim: To evaluate the performance of Quantamatrix's dRAST v2.5 system (Seoul, South Korea) for AST directly from positive blood cultures as compared to the Disk-Diffusion (DD) and VITEK 2 methods., Methods: The study included 191 positive blood cultures from clinical samples and spiked blood culture bottles. Following Gram staining and species-level identification, AST was performed by VITEK 2 and standard DD methods using CLSI (2021) interpretation., Results: dRAST demonstrated very good AST performance for a Gram-negative isolate, and good performance for Gram-positive isolates, meeting CLSI criteria for the acceptance of a new method. Antimicrobials that were not considered verified compared to VITEK 2 and DD were cefazolin, ceftazidime, meropenem, and trimethoprim/sulfamethoxazole for Gram-negatives and clindamycin, erythromycin, penicillin, and oxacillin for Gram-positives. dRAST ESBL detection results were strongly correlated with the ESBL phenotypes obtained with other methods. Additional resistance mechanisms were in concordance with traditional tests., Conclusions: dRAST demonstrated good AST performance, meeting CLSI criteria for most relevant antibiotics. dRAST was associated with a significant reduction in time-to-results, labor, and the subjectivity of result analyses, making it a valuable addition to efforts supporting the treatment of patients with bacteremia. AST (antimicrobial susceptibility test), blood culture, dRAST, rapid methods, sepsis, turnaround time (TAT)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Antibiotic susceptibility tests (AST) done on blood cultures are critical for the treatment of patients suspected to be suffering from bloodstream infection. The objective of this study was to evaluate the accuracy and reliability of disc diffusion AST conducted directly (direct AST) from flagged-positive blood cultures, especially for Gram-positive cocci bacteria., Methodology: This study compared direct AST with conventional AST (broth micro-dilution). The antibiotics studied were piperacillin/tazobactam, gentamicin, ceftazidime, erythromycin, and penicillin. Accuracy was determined by calculating very major, major, and minor errors. The reliability was determined by categorical agreement and weighted Kappa index., Results: Gram-positive cocci bacteria were grown in pairs of blood culture bottles and tested with the two methods of AST. No very major errors were detected among the five types of antibiotics. Major errors of 2.56% and minor errors of 4.93% were found when testing gentamicin. The major and minor errors when testing erythromycin were 2.85% and 1.23%, respectively. Perfect agreements (categorical agreement: 100%; weighted Kappa index: 1) of the two AST methods were observed with piperacillin/tazobactam, ceftazidime, and penicillin. Almost perfect agreement was found with gentamicin and erythromycin. Categorical agreement results when testing antibiotics gentamicin and erythromycin were 93.83% and 97.53%, respectively. In addition, the weighted-Kappa index when testing these two antibiotics were 0.92 and 0.96, respectively., Conclusions: The accuracy and reliability of the direct AST was within acceptable limits., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2024 Osman Sianipar, Rizka N Firdaus, Ira Puspitawati.)

The use of rapid disk diffusion or modified automated antimicrobial susceptibility testing (AST) system approaches demonstrates excellent performance for gram-negative organisms directly from blood cultures. In a recent study, S. Khan, A. Das, A. Mishra, A. Vidyarthi, et al. (Microbiol Spectr 12:e03081-23, 2024, https://doi.org/10.1128/spectrum.03081-23) compared the performance of three direct-from-blood AST methods against standard of care disk diffusion and automated AST. The results demonstrated high categorical agreements and low error rates across three protocols. The study suggests that locally validated direct-from-blood AST protocols offer reliable and fast results, particularly for resource-limited settings. However, local context and workflows should be considered prior to implementing rapid AST protocols, and more research is needed on the performance of rapid AST protocols for gram-positive organisms., Competing Interests: Andrea Prinzi is a paid employee of bioMerieux (Field Medical Director, US Medical Affairs).

Introduction. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) specifies the agar depth (4±0.5 mm) when performing antimicrobial susceptibility testing (AST). Since the infrastructure to produce standardized agar may be lacking in settings with limited resources, we wanted to examine to what extent variation in agar depth affects the inhibition zone diameters of quality control (QC) strains and AST of clinical isolates. Methods. The inhibition zone diameters on Mueller-Hinton II agar with different depths (2-6 mm) were tested for various QC strain-antimicrobial agent combinations using Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. The relationship between zone diameters at different agar depths and MICs was investigated for 35 clinical isolates ( E. coli , Klebsiella pneumoniae , S. aureus and P. aeruginosa ) from Sierra Leone using MICs as the reference. Results. The inhibition zone diameters were within the acceptance ranges as defined by the EUCAST for the majority of QC strains and antimicrobials, independent of the agar depth. At extreme agar depths, inhibition zones were more frequently out of range. The accuracy of AST varied for clinical isolates at different agar depths for categorical agreement (85.8-94.6%), major error rate (0.4-2.1%) and very major error rate (VME: 3.3-12.5%). Conclusions. Even if the QC strains were in the acceptance range at different agar depths, this does not rule out unacceptably high VME rates (>3%) in clinical isolates.

To evaluate the in vitro activity of ampicillin-sulbactam and cefoperazone-sulbactam against A. baumannii using the broth disk elution testing, a total of 150 A. baumannii isolates were collected from across China between January 2019 and January 2021, including 51 carbapenem-susceptible and 99 carbapenem-resistant isolates. Broth disk elution (BDE) and the broth microdilution (BMD) method were performed for all strains. The concentration range of the BDE was 10/10 µg/mL, 20/20 µg/mL, and 30/30 µg/mL for ampicillin-sulbactam, and 37.5/15 µg/mL, 75/30 µg/mL, 112.5/45 µg/mL, and 150/60 µg/mL for cefoperazone-sulbactam, respectively. Compared with BMD, the BDE results of ampicillin-sulbactam and cefoperazone-sulbactam showed a categorical agreement of 83.3% (125/150) and 95.3% (143/150), with minor errors of 16.7% (25/150) and 4.7% (7/150), respectively. No major error or very major errors were detected. The sensitivity differences by BDE of carbapenem-resistant A. baumannii (CRAb) to different concentrations of ampicillin-sulbactam showed statistically significant (p < 0.017), while those to cefoperazone-sulbactam at 37.5/15 µg/mL, 75/30 µg/mL, and 112.5/45 µg/mL were significant (p < 0.008). However, no significant difference in sensitivity was observed between 112.5/45 µg/mL and 150/60 µg/mL (p > 0.008). In conclusion, the BDE is a reliable and convenient method to detect the in vitro activity of cefoperazone-sulbactam against A. baumannii, and the results could serve as a clinical reference value when deciding whether or not to use high-dose sulbactam for the treatment of A. baumannii infections., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The escalation of antimicrobial resistance (AMR) due to the excessive and inappropriate use of antimicrobials has prompted the urgent need for more rapid and effective antimicrobial susceptibility testing (AST) methods. Conventional AST techniques often take 16-24 h, leading to empirical prescription practices and the potential emergence of AMR. The study aimed to develop a rapid disk diffusion (RDD) method utilizing laser speckle formation (LSF) technology to expedite AST results. The study aimed to evaluate the performance of LSF technology in determining antimicrobial susceptibility. In this study, preclinical and clinical settings were established to compare the LSF technology with conventional disk diffusion (DD) methods to measure the inhibition zones. Preclinical experiments with different bacterial strains demonstrated more than 70% categorical agreement (CA) against most antimicrobials. Further, clinical experiments with multiple strains and antibiotics revealed CA ranging from 40 to 79%, while major and minor discrepancies were observed around 30% and 11%, respectively. These observations revealed high concordance between RDD and DD for multiple antimicrobials in multiple species. The results underscore the potential of RDD-based LSF technology for hastening AST procedures. The current study is marked by a unique equipment setup and analysis approach. Collectively, the suggested laser-based RDD showed greater potential than previously developed comparable methods. The proposed method and design have a higher application potential than formerly developed similar technologies. Together, the study contributes to the ongoing development of rapid AST methods., (© 2024. The Author(s).)

Objectives: To investigate the prevalence of ampicillin resistance in Haemophilus influenzae and the diagnostic accuracy of the EUCAST recommended disc diffusion method to detect the increasingly prevalent ampicillin resistance due to the presence of PBP3 alterations based on mutations in the ftsI gene., Methods: During a 6-month period all consecutive non-duplicate H. influenzae isolates were prospectively collected and stored. MICs of ampicillin were determined by broth microdilution (BMD). PCR was performed to detect mutations in the ftsI gene. Results of routine disc diffusion susceptibility testing, including the penicillin screening test in accordance with the current EUCAST methodology, as well as additional Etest results, were compared to the BMD as the reference method., Results: In 102 isolates, the prevalence of ampicillin resistance was 28% (29/102) by BMD. There was a good correlation between MICs of ampicillin and the presence of a β-lactamase and/or an ftsI gene mutation. The prevalence of ampicillin resistance was overestimated using the EUCAST method (33% (34/102)) and underestimated when an additional Etest was used (24% (24/102)) (not significant). The sensitivity and specificity of the EUCAST methodology for the detection of ampicillin resistance were 97% ((28/29); 95% CI, 82-100%) and 92% ((67/73); 95% CI, 83-97%), respectively., Conclusions: The prevalence of ampicillin resistance was 28%, as determined by BMD. Although the overall diagnostic accuracy of the EUCAST ampicillin disc diffusion was high, misclassification of ampicillin susceptibility may still occur., (Copyright © 2024 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Purpose: To assess performance of Etest®, Vitek®2 and BD Phoenix™ to determine the susceptibility of Streptococcus pneumoniae strains to penicillin, ampicillin and cefotaxime., Methods: Sixty unique S. pneumoniae challenge strains were selected to cover a wide range of penicillin, ampicillin and cefotaxime minimal inhibitory concentrations (MICs). Strains were analyzed in four different Belgian laboratories. Etest® benzylpenicillin (BEN), ampicillin/amoxicillin (AMP) and cefotaxime (CTA) (bioMérieux), Vitek®2 AST-ST03 (bioMérieux) and BD Phoenix™ SMIC/ID-11 testing were each performed in two different labs. Results were compared to Sensititre® broth microdilution (BMD) (Thermo Fisher Scientific) results. MIC results were interpreted using EUCAST non-meningitis breakpoints (v 13.0)., Results: Essential agreement (EA) was ≥ 90% for all methods compared to BMD, except for Etest® BEN on Oxoid plate (58.3%), Etest® AMP (both on Oxoid (65.8%) and BD BBL plate (84.2%)). Categorical agreement (CA) for penicillin was only ≥ 90% for Vitek®2, for other methods CA ranged between 74 and 84%. CA for AMP was for all methods < 90% (range 75.8-88.3%) and CA for CTA was between 87 and 90% for all methods except for Etest on Oxoid plate (79.2%)., Conclusions: Our study indicates that Vitek®2 and BD Phoenix™ are reliable for providing accurate pneumococcal susceptibility results for BEN, AMP and CTA. Using Etest BEN or AMP on Oxoid plate carries a risk of underestimating the MIC and should be interpreted with caution, especially when the obtained MIC is 1 or 2 doubling dilutions below the S or R clinical breakpoint., (© 2024. The Author(s).)

There are increasing reports of carbapenem-resistant Enterobacterales (CRE) that test as cefepime-susceptible (S) or susceptible-dose dependent (SDD). However, there are no data to compare the cefepime testing performance of BD Phoenix automated susceptibility system (BD Phoenix) and disk diffusion (DD) relative to reference broth microdilution (BMD) against carbapenemase-producing (CP bla KPC -CRE) and non-producing (non-CP CRE) isolates. Cefepime susceptibility results were interpreted according to CLSI M100Ed32. Essential agreement (EA), categorical agreement (CA), minor errors (miEs), major errors (MEs), and very major errors (VMEs) were calculated for BD Phoenix (NMIC-306 Gram-negative panel) and DD relative to BMD. Correlates were also analyzed by the error rate-bounded method. EA and CA for CP bla KPC -CRE isolates ( n = 64) were <90% with BD Phoenix while among non-CP CRE isolates ( n = 58), EA and CA were 96.6%, and 79.3%, respectively. CA was <90% with DD for both cohorts. No ME or VME was observed for either isolate cohort; however, miEs were >10% for CP bla KPC -CRE and non-CP CRE with BD Phoenix and DD tests. For error rate-bounded method, miEs were <40% for I High + 1 to I Low - 1 ranges for CP bla KPC -CRE and non-CP CRE with BD Phoenix. Regarding disk diffusion, miEs were unacceptable for all MIC ranges among CP bla KPC -CRE. For non-CP CRE isolates, only I High + 1 to I Low - 1 range was acceptable at 37.2%. Using this challenge set of genotypic-phenotypic discordant CRE, the BD Phoenix MICs and DD susceptibility results trended higher (toward SDD and resistant phenotypes) relative to reference BMD results yielding lower CA. These results were more prominent among CP bla KPC -CRE than non-CP CRE., Competing Interests: P.J.S. reports grants and personal fees from OpGen, Inc., bioMérieux, Inc., Qiagen, and BD Diagnostics; grants from Affinity Biosensors and T2 Biosystems; and personal fees from Shionogi, Inc., Entasis, Merck, and GeneCapture, outside the submitted work. D.P.N. served as a consultant or speaker's bureau member or has received research funding from Allergan, Cepheid, Merck, Pfizer, Wockhardt, Shionogi, Tetraphase, and Venatorx. T.E.A. has received research funding from Venatorx, Paratek, Shionogi, and Spero Therapeutics. A.F. has no conflicts of interest to declare.

Objectives: To evaluate the performance of an in-house developed disk diffusion method for aztreonam in combination with avibactam against Enterobacteriales., Methods: The in vitro antibacterial activity of aztreonam with avibactam against 204 carbapenemase-producing Enterobacteriales was determined by a disk diffusion method, with a broth microdilution method as a reference., Results: The optimal S/R breakpoints for disk diffusion tests of 30/20 and 10/4 µg disks, calculated by the dBETs software using the model-based approaches, were ≥22/≤21 and ≥12/≤11 mm, respectively. On the basis of the estimated breakpoints, the CAs for disk diffusion tests of 30/20 and 10/4 µg aztreonam/avibactam disks were both 98.0%, with 0.5% major error and 37.5% very major error., Conclusions: The home-made disk diffusion method is an economical and practical method for clinical microbiology laboratories to determine the antibacterial susceptibility of aztreonam with avibactam against Enterobacteriales., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Antimicrobial susceptibility testing (AST) by disk diffusion provides an accurate image of bacterial growth, enabling the detection of culture purity, heterogeneous growth, and antibiotic interactions. However, this manual method is time-consuming and visual interpretation is prone to errors. To overcome these disadvantages, the Radian® In-Line Carousel (Copan, Brescia, Italy) was launched, which is a WASPLab® module dedicated to full automation of (pre)-analytical steps as well as interpretation of disk diffusion AST. However, until now, no evaluation of Radian® against manual disk diffusion has been performed. We assessed the categorical agreement (CA) between standardized disk diffusion (reference method) and Radian® using EUCAST 2021 breakpoints. We tested 135 non-duplicate strains, selected from the National EUCAST challenge panel, clinical strains, and external quality controls. The strains included Enterobacterales (n = 63), Enterococcus faecalis (n = 3), Enterococcus faecium (n = 10), Pseudomonas aeruginosa (n = 16), Staphylococcus aureus (n = 19), coagulase-negative staphylococci (n = 4), and Streptococcus spp. (n = 20). Furthermore, we explored antibiotic disk thermolability in the WASP Radian® carousel by testing 10 ATCC® strains up to 7 days. The observed CA was 95.3%, 96.3%, 93.8%, 97.3% and 98.0% for Enterobacterales, Enterococcus spp., P. aeruginosa, Staphylococcus spp. and Streptococcus spp., respectively, resulting in an acceptable overall CA for all groups. (Very) major error rates were ≤ 5% for all antibiotics. Antibiotic disk thermostability was confirmed up to 4 days in the WASP Radian® In-Line Carousel. The Radian® In-Line Carousel provides a fully automated solution for accurate disk diffusion AST, reducing workload and improving standardization and traceability. In addition, our study demonstrated the thermostability of antibiotic disks up to 4 days in the WASP Radian® In-Line Carousel., (© 2024. The Author(s).)

Purpose: The study explores the impact of significant interpretative breakpoint changes for aminoglycosides and piperacillin-tazobactam in Enterobacterales and Pseudomonas aeruginosa, considering PK/PD, clinical data, and susceptibility on clinical reporting and use., Procedure: Between January 2021 and June 2023, a total of 189,583 samples were processed for bacterial pathogens and antimicrobial susceptibility testing was performed using disc diffusion method/VITEK® 2 Compact system/broth microdilution. WHONET software was utilised to capture and analyse the changes in the interpretation of disc diffusion method, following updates to CLSI M100 documents in comparison to previous editions. Antimicrobial consumption data was collected and interpreted as DDD/100 bed days using AMC tool software. Here, we present data for 13,615 members of Order Enterobacterales and 1793 Pseudomonas aeruginosa isolates., Finding: Enterobacterales exhibited a significant susceptibility drop of 14.7% for gentamicin and 21.7% for amikacin. Pseudomonas aeruginosa showed an increase in isolates with intermediate tobramycin susceptibility, from 0.6% to 29.7%, with relatively minor changes in piperacillin-tazobactam interpretation., Conclusion: The changes indicate a shift toward increased 'resistance' and 'intermediate susceptibility' for these antibiotics, emphasizing the need for cautious use and leveraging PK/PD knowledge for improved antibiotic utilization, patient outcomes, and antimicrobial stewardship., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Antibiotic resistance causes a major threat to patients suffering from infectious diseases. Accurate and timely assessment of Antibiotic Susceptibility Test (AST) is of great importance to ensure adequate treatment for patients and for epidemiological monitoring. Disc Diffusion Test (DDT) is a standard and widely used method for AST. Manual interpretation of DDT results is a tedious task and susceptible to human errors. Computer vision-based automated interpretation of DDT results will speed up the process and reduces the manpower requirement. This would assist the physician to initiate the antibiotic treatment for the patients on time and results in saving the patient's life. The crucial step in automatic interpretation of DDT result is to measure and present the diameter of zone of inhibition without manual intervention. The existing methods require manual interventions at various stages during inhibition zone diameter measurement for some typical cases. This issue is addressed in the present work through maximally stable extremal regions (MSER) based algorithm. Dataset consisting of 60 agar plate images that includes different agar medium, images having different resolution and visual quality is used to validate the proposed method. Experimental results demonstrated that there is a strong correlation between standard method and the proposed method.

Objectives: The aim of this laboratory-based study was to compare carbapenem MICs yielded by Sensititre, Vitek 2, MicroScan WalkAway plus and Etest for Oxacillin (OXA)-48-like Klebsiella pneumoniae isolates., Methods: Analysis was performed for categorical agreement for ertapenem, meropenem, and imipenem, and the proportion of isolates with MICs ≤8μg/mL and the MIC50/MIC90 for meropenem and imipenem, from a convenience sample of 82 deduplicated blood culture OXA-48-like K. pneumoniae isolates., Results: The proportion of isolates testing susceptible to ertapenem by Etest (19/82, 23.1%) differed from Sensititre/Vitek (0/82) and MicroScan (2/82, 2.4%) (p < 0.001 for all). For meropenem, the proportion of isolates susceptible by Etest (31/82, 37.8%) differed from Sensititre/Vitek (16/82, 19.5%) (p = 0.015). There was variation in the proportion of isolates that tested imipenem susceptible when comparing Sensititre (9/82, 11%) and Vitek (8/82, 9.8%) to MicroScan (27/82, 32.9%), p = 0.001 and p < 0.001, respectively, Sensititre and Vitek to Etest (45/82, 54.9%), p < 0.001 for both, and MicroScan to Etest, p = 0.007. The proportion of isolates with meropenem MICs ≤8μg/mL with Sensititre and Vitek differed significantly from Etest, 58.5% and 85.4%, respectively, p < 0.001. A 2-fold difference between the Sensititre and Vitek meropenem and imipenem MIC at which ≥50% of isolates were inhibited compared to the MicroScan, and a 4-fold difference compared to Etest, was present., Conclusions: Substantial variability in carbapenem MICs for OXA-48-like carbapenemase-producing Enterobacterales isolates by the four methods was demonstrated. Performance characteristics verification of MIC methods in use for the predominant carbapenemase-producing Enterobacterales type is required by laboratories to optimize the accuracy of carbapenem reporting., (Copyright © 2022 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objectives: To evaluate the performance of two rapid antimicrobial susceptibility testing (RAST) methods to determine ceftazidime/avibactam susceptibility directly from blood cultures (BCs)., Methods: A total of 246 Escherichia coli or Klebsiella pneumoniae isolates were tested for ceftazidime/avibactam susceptibility directly from BC bottles using EUCAST RAST and Etest® RAST. Results obtained after 4, 6 and 8 h of incubation were compared with those obtained by reference broth microdilution on pure overnight subcultures., Results: In total, the proportion of readable zones after 4 h of incubation was 96.7% and reached 100% after 6 and 8 h of incubation. EUCAST RAST yielded >98% of categorical agreement (CA) with all reading times. Major error (ME) and very major error (VME) rates were inferior to 3%, for each of the reading times. The proportion of results in the area of technical uncertainty (ATU) was almost similar (3.8%-4.1%) at the different reading times. DET-RAST yielded 97.5%, 98% and 99.6% of CA with readings at 4, 6 and 8 h, respectively. One (0.6%) ME was observed at each reading time, whereas five (5.9%) and four (4.5%) VMEs were observed analysing readings at 4 and 6 h, respectively. No VME was observed with readings at 8 h., Conclusions: EUCAST RAST was accurate to determine ceftazidime/avibactam susceptibility of carbapenemase-producing K. pneumoniae and E. coli directly from BC bottles. DET-RAST has the advantage of determining MIC values and avoiding ATU results but showed to be an accurate method only with reading at 8 h., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Disk diffusion is a slow but reliable standard method for measuring the antimicrobial susceptibility of microorganisms. Our objective was to improve the turnaround time for this method by reducing the time that cultures are incubated before setting up disk diffusion testing. For initial method development, clinical isolates ( n  = 13) and quality control strains ( n  = 8) of bacteria were inoculated on blood agar and were incubated at 35°C for either 6, 10, or 24 h before performing disk diffusion testing, in triplicate, using a panel of clinically appropriate antimicrobial agents. Disk diffusion zone sizes were interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines. Compared to standard 24 h of incubation, early 6-h growth had 1.3% major errors (MEs) and 1.9% very major errors (VMEs), whereas 10-h growth yielded 0.7% MEs and no VMEs. Categorical agreement with standard incubation was similar for both 6 h (96.7%) and 10 h (96.7%) growth. Inhibitory zone size from 6 h ( r 2 = 0.98) and 10 h ( r 2 = 0.99) growth correlated well with results from standard conditions. Based on these results, we performed disk diffusion under optimized conditions (6 h growth), using 100 additional clinical isolates, demonstrating a high level of categorical agreement (917 of 950 measurements [96.5%]; 95% confidence interval [CI], 95.2 to 97.5%), as well as no VMEs or MEs. Using early growth for disk diffusion testing is a simple and accurate method for susceptibility testing that can reduce time to results by as much as 18 h, compared to standard incubation, with no additional supply costs or equipment/instrumentation.

Helicobacter pylori is an important human pathogen associated with peptic ulcer disease, dyspepsia, and gastric malignancy. Antimicrobial susceptibility testing (AST) is often requested for patients who fail eradication therapy. The Clinical and Laboratory Standards Institute (CLSI) reference method, agar dilution (AD), is not performed in most laboratories and maintaining organism viability during transit to a reference laboratory is difficult. We assessed the performance of the Etest (bioMérieux) as a method for H. pylori AST in comparison to AD. Etest MICs were determined for 83 H. pylori isolates at ARUP and Cleveland Clinic (CC). Categorical agreement (CA), very major, major, and minor errors (VME, ME, and mE) were determined for Etest using AD performed at Mayo Clinic Laboratories as the reference method. Testing on isolates with errors was repeated to determine final results summarized below. For clarithromycin, 66.3% of isolates were resistant (R) by AD; Etest results at each laboratory showed 1mE (1.2%) and 1 ME (3.8%). For tetracycline, only 2 isolates were R by AD; a single VME occurred at both sites (98.8% CA, 50% VME) with the same isolate. Applying EUCAST levofloxacin breakpoints to interpret ciprofloxacin results, 60.2% of isolates were R by AD; ARUP CA was 97.6% (1 ME (3%), 1 VME (2%)) and CC CA was 96.3% (1 ME (3%), 2 VMEs (4%)). Despite high error rates, the categorical agreement was acceptable (>90%) for all three antibiotics between AD and Etest. In-house susceptibility testing by gradient diffusion can allow for testing of fastidious organisms that may not survive transport to specialized laboratories; however, the method is not without technical challenges. Characterization of resistance mechanisms, increased AD dilutions, and testing from the same inoculum may determine if the observed errors reflect technical issues or breakpoints that need optimization. IMPORTANCE Routine antimicrobial susceptibility testing (AST) of Helicobacter pylori by agar dilution is difficult to perform and not practical in most clinical microbiology laboratories. The Etest gradient diffusion method can be a reliable alternative for H. pylori AST with the advantage of being a less laborious quantitative method. This work reveals that an optimized Etest method can provide acceptable performance for H. pylori AST and describes the challenges associated with this methodology.

The objective of this research was to evaluate the correlation between inhibitory zones and MIC when testing ceftazidime-avibactam using disk diffusion, Etest, and broth microdilution method established by the Clinical and Laboratory Standards Institute (CLSI). Four-hundred and 58 isolates of Enterobacterales isolated from 54 medical centers from the China Antimicrobial Surveillance Network (CHINET) in 2016 to 2020 were collected. Antimicrobial susceptibility testing using broth microdilution, Etest, and disk diffusion were performed according to the CLSI. Of the 458 Enterobacterales , 17.2% (79/458) and 82.8%(379/458) were resistant or susceptible to ceftazidime-avibactam by broth microdilution, respectively. Compared with the broth microdilution method, the categorical agreement (CA) and essential agreement (EA) of the Etest were 99.6% (456/458) and 94.8% (434/458), respectively; the major error (ME) and very major error (VME) were both 0.2% (1/458). For disk diffusion, the CA and VME were 99.8% (457/458) and 0.2% (1/458), respectively. For Escherichia coli, the CA and EA of the Etest were 100% and 97.1% (135/139), respectively. The CA of the disk diffusion was 100%. For Klebsiella pneumoniae, the CA and EA of the Etest were 99.3% (288/290) and 93.4% (271/290), respectively, the ME and VME were both 0.3% (1/290). The CA and VME of disk diffusion were 99.7% (289/290) and 0.3% (1/290), respectively. For other Enterobacterales , the CA and EA of the Etest were 100% and 96.6% (28/29), respectively. The CA of the disk diffusion was 100%. Ceftazidime-avibactam disk diffusion (30/20-μg disks) and Etest demonstrated good performance for ceftazidime-avibactam susceptibility testing against Enterobacterales clinical isolates. IMPORTANCE Multidrug-resistant Gram-negative bacteria, especially for extended-spectrum β-lactamases-producing and carbapenemase-producing Enterobacterales , are disseminating rapidly around the world. Treatment options for these infections are limited, which prompt the development of novel or combinational therapies to combat the infections caused by multidrug-resistant pathogens. The newly available β-lactam combination agent ceftazidime-avibactam has been demonstrated good in vitro and in vivo activity against ESBL, AmpC, KPC-2, or OXA-48-like-producing isolates and has shown promise in treating carbapenem-resistant Enterobacterales infections. Concerningly, there are few available automated systems for ceftazidime-avibactam susceptibility testing, and the broth microdilution method is hard to perform in most routine laboratories. Therefore, we urgently need an economical and practical method for the accurate detection of ceftazidime-avibactam activity against Gram-negative bacilli. Here, we evaluate the performance of the disk diffusion and Etest compared with the reference broth microdilution method against Enterobacterales clinical strains.

On the first of January 2019, the European Committee on Antimicrobial Susceptibility Testing, EUCAST, introduced the concept of "area of technical uncertainty" (ATU). The aim was to report on the incidence of ATU test results in a selection of common bacterial species and the subsequent impact on antimicrobial resistance categorization and workload. A retrospective analysis of clinical samples collected from February 2019 until November 2019 was performed. Susceptibility to amoxicillin-clavulanic acid and piperacillin-tazobactam in Enterobacterales (Escherichia spp., Klebsiella spp., Proteus spp.), piperacillin-tazobactam in Pseudomonas aeruginosa, and amoxicillin-clavulanic acid and cefuroxime in Haemophilus influenzae was studied. Disk diffusion antibiotic susceptibility testing was read and interpreted by ADAGIO 93400 automated system (Bio-Rad, France). In case of an inhibition zone in the ATU, strains were retested using gradient minimal inhibitory concentration method (Etest, BioMérieux, France). Overall, 14,164 isolate-antibiotic combinations were tested in 7922 isolates, resulting in 1204 (8.5%) disk zone diameters in the ATU region. Retesting of ATUs with Etest resulted in a category change from S to R for amoxicillin-clavulanic acid in 63/498 (12.7%) of Escherichia spp., 2/58 (3.4%) of Klebsiella spp., 2/37 (5.4%) of Proteus spp., and 6/125 (4.8%) of Haemophilus influenzae. For piperacillin-tazobactam, a category change from S to R was found in 33/92 (35.9%) of Pseudomonas aeruginosa. We conclude that ATU testing has a substantial impact on the correct interpretation of antimicrobial resistance, at the expense of turn-around time and with the cost of additional workload., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Plazomicin (PLZ), brand name ZEMDRI (Cipla Therapeutics), is a novel aminoglycoside antibiotic approved by the U.S. Food and Drug Administration (FDA) for treatment of complicated urinary tract infections including pyelonephritis. ETEST® is a gradient diffusion method that represents an alternative to the more laborious broth micro-dilution (BMD) method for performing antimicrobial susceptibility testing (AST). A multi-center evaluation of the performance of the new ETEST PLZ (bioMérieux) was conducted in comparison with BMD following FDA and International Standards Organization (ISO) recommendations using FDA-defined breakpoints. Clinical isolates of Enterobacterales ( n  = 598) were included. Fifty-three isolates were resistant to PLZ according to BMD. Overall, the ETEST PLZ demonstrated 99.0% essential agreement (EA), 92.8% category agreement (CA), 1.9% very major errors (VME), 0% major errors (ME), and 7.0% minor errors (mE) with both clinical and challenge isolates of Enterobacterales. The VME was found for a single Serratia marcescens strain. Individual species demonstrated EA rates ≥ 90%. In conclusion, we report that ETEST PLZ represents an accurate tool for performing PLZ AST of Enterobacterales.

This work addresses the mathematical model building to detect the diameter of the inhibition zone of gilaburu (Viburnum opulus L.) extract against eight different Fusarium strains isolated from diseased potato tubers. Gilaburu extracts were obtained with acetone, ethanol or methanol. The isolated Fusarium strains were: F. solani, F. oxysporum, F. sambucinum, F. graminearum, F. coeruleum, F. sulphureum, F. auneaceum and F. culmorum. In general, it was observed that ethanolic extracts showed highest antifungal activity. The antifungal activity of extracts was evaluated with machine learning (ML) methods. Several ML methods (classification and regression trees (CART), support vector machines (SVM), k-Nearest Neighbors (k-NN), artificial neural network (ANN), ensemble algorithms (EA), AdaBoost (AB) algorithm, gradient boosting (GBM) algorithm, random forests (RF) bagging algorithm and extra trees (ET)) were applied and compared for modeling fungal growth. From this research, it is clear that ML methods have the lowest error level. As a result, ML methods are reliable, fast, and cheap tools for predicting the antifungal activity of gilaburu extracts. These encouraging results will attract more research efforts to implement ML into the field of food microbiology instead of traditional methods., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Current methods for screening small molecules that inhibit the plasmid pCD1-encoded Yersinia pestis type III secretion system (T3SS) include lengthy growth curves followed by multistep luminescence assays or Western blot assays to detect secretion, or lack thereof, of effector proteins. The goal of this research was to develop a novel disk diffusion assay on magnesium oxalate (MOX) agar as a simple way to evaluate the susceptibility of Y. pestis to type III secretion system inhibitors. MOX agar produces distinct Y. pestis growth characteristics based on the bacteria's ability or inability to secrete effector proteins; small, barely visible colonies are observed when secretion is activated versus larger, readily visible colonies when secretion is inhibited. Wild-type Y. pestis was diluted and spread onto a MOX agar plate. Disks containing 20 μl of various concentrations of imidocarb dipropionate, a known Y. pestis T3SS inhibitor, or distilled water (dH 2 O) were placed on the plate. After incubation at 37°C for 48 h, visible colonies of Y. pestis were observed surrounding the disks with imidocarb dipropionate, suggesting that T3S was inhibited. The diameter of the growth of colonies surrounding the disks increased as the concentration of the T3SS inhibitor increased. Imidocarb dipropionate was also able to inhibit Y. pestis strains lacking effector Yops and Yop chaperones, suggesting that they are not necessary for T3S inhibition. This disk diffusion assay is a feasible and useful method for testing the susceptibility of Y. pestis to type III secretion system inhibitors and has the potential to be used in a clinical setting. IMPORTANCE Disk diffusion assays have traditionally been used as a simple and effective way to screen compounds for antibacterial activity and to determine the susceptibility of pathogens to antibiotics; however, they are limited to detecting growth inhibition only. Consequently, antimicrobial agents that inhibit virulence factors, but not growth, would not be detected. Therefore, we developed a disk diffusion assay that could detect inhibition of bacterial virulence factors, specifically, type III secretion systems (T3SSs), needle-like structures used by several pathogenic bacteria to inject host cells with effector proteins and cause disease. We demonstrate that magnesium oxalate (MOX) agar can be used in a disk diffusion assay to detect inhibition of the T3SS of Yersinia pestis, the causative agent of bubonic plague, by small-molecule inhibitors. This assay may be useful for screening additional small molecules that target bacterial T3SSs or testing the susceptibility of patient-derived samples to drugs that target T3SSs.

Objectives: The emergence of multidrug-resistant Neisseria gonorrhoeae (NG) is a major global health threat necessitating response and control measures. NG antimicrobial resistance (AMR) surveillance data from sub-Saharan countries is exceedingly limited. This paper aims to describe the establishment, design and implementation of a standardised and quality-assured gonococcal surveillance programme and to describe the susceptibility patterns of the cultured gonococcal isolates in Kampala, Uganda., Methods: From March 2018 to September 2019, using the WHO Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP) protocol, consecutive males with urethral discharge syndrome were recruited from 10 surveillance sites in Kampala City, Uganda, in collaboration with the Ministry of Health. Males completed a questionnaire and provided a urethral swab specimen. Culture, identification and antimicrobial susceptibility testing (Etest) were performed., Results: Of the 1013 males recruited, 73.1% (740/1013) had a positive Gram stain and 51.1% (n=518) were culture-positive for NG. Using Etest (458 isolates), the resistance to ciprofloxacin was 99.6%. Most isolates were susceptible to azithromycin, cefoxitin and gentamicin, that is, 99.8%, 98.5% and 92.4%, respectively, and all isolates were susceptible to ceftriaxone and cefixime., Conclusions: We established a standardised, quality-assured WHO EGASP. Using Etest, 458 isolates were characterised, with associated epidemiological surveillance data, in 1.5 years, which by far exceed the minimum 100 isolates per year and country requested in the WHO Global GASP, to detect AMR levels with confidence. These isolates with the epidemiological data can be used to develop population level interventions., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Tigecycline is an alternative antibiotic for managing carbapenem-resistant Gram-negative bacterial infections. However, disk diffusion and automated testing often show false-intermediate or false-resistant results in tigecycline susceptibility, misleading clinical antimicrobial therapy. Broth microdilution (BMD) is the reference method for testing tigecycline susceptibility, but it is labor intensive and time consuming to perform in clinical laboratories. Therefore, a simple and accurate method is urgently needed. We evaluated the performance of VITEK 2, E-test, Kirby-Bauer disk diffusion (KB), and modified KB disk diffusion (mKB) versus BMD in testing tigecycline susceptibility of 372 strains of carbapenem-resistant Klebsiella pneumoniae (CRKP) and 346 strains of carbapenem-resistant Acinetobacter baumannii (CRAB). BMD confirmed that 96.8% of CRKP and 91% of CRAB strains were susceptible to tigecycline. E-test, VITEK 2, KB, and mKB yielded categorical agreement of 96.7/59.3%, 69.9/54.3%, 78.5/87.3%, and 96.5%/91% for CRKP/CRAB, respectively. No very major error was found for either CRKP or CRAB by any method. No major error was found for CRKP or CRAB by the mKB method. The mKB method enhanced by R-buffer is simple, accurate, and inexpensive for clinical laboratories to test the susceptibility of CRKP and CRAB isolates to tigecycline.

Culture-based antimicrobial susceptibility testing is a crucial method for the management of Helicobacter pylori infection . It must follow the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations for fastidious microaerophilic bacteria, with some possible variation especially for the medium to be used. It is recommended to test six antibiotics by diffusion using strips charged with an antibiotic gradient in order to determine the minimum inhibitory concentrations (MICs). Two of these antibiotics, clarithromycin and levofloxacin, are more important because of frequent resistance which jeopardizes the success of the treatment.

Background: Considering the interesting role in the peptidoglycan biosynthesis pathway, the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase is an attractive target to develop new antibacterial agents. It catalyzes the first key step of this pathway and its inhibition leads to bacterial cell death. Fosfomycin is known as the natural inhibitor of MurA., Objective: The study aimed to introduce new inhibitors of MurA by virtual screening of different chemical compounds libraries, and test the best scored "virtual hits" against three pathogenic bacteria: Escherichia coli, Bacillus subtilis and Staphylococcus aureus., Methods: A virtual screening of the structural analogues of fosfomycin downloaded from the Pub- Chem database was performed. Moreover, French National Chemical Library and ZINC database were also utilized to identify new structures different from fosfomycin. FlexX was the software used for this study. The antibacterial testing was divided into two methods: disk diffusion and broth dilution., Results: A set of virtual hits was found to have better energy score than that of fosfomycin, seven of them were tested in vitro. In addition, the disk diffusion method explored four compounds that exhibited antibacterial activity: CID-21680357 (fosfomycin analogue), AB-00005001, ZINC04658565, and ZINC901335. The testing was continued by broth dilution method for both compounds CID-21680357 and ZINC901335 to determine their minimum inhibitory concentrations, and ZINC901335 had the best value with 457μg/ml against Staphylococcus aureus., Conclusion: Four compounds were found and proven in silico and in vitro to have antibacterial activity, namely CID-21680357, AB-00005001, ZINC04658565, and ZINC901335., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Over the past decade, drug resistance pattern has worsened for many of the uropathogens due to overuse of antibiotics for empiric treatment. The burden of extended spectrum beta-lactamase (ESBL) producing Enterobacteriaceae associated urinary tract infections (UTI) has become increasingly more common, limiting treatment options among children presenting with febrile UTI. We investigated the burden and correlates of ESBL producing Enterobacteriaceae associated UTI among children and antibacterial resistance pattern., Methods: 284 midstream urine specimens were collected using standard aseptic techniques from 284 children who were diagnosed with suspected UTI. Urine culture and bacteria isolation were performed following standard bacteriological techniques. The Kirby-Bauer disk diffusion technique and the double-disc synergy test were used to investigate antibiotic susceptibility and presence of ESBL production., Results: UTI was confirmed using a positive urine culture for a relevant pathogen in 96/284 (33.8%) of the cases. Enterobacteriaceae accounted for 75% (72/96) of etiologies of UTI in children. The most frequent Enterobacteriaceae spp. were E. coli , 44.4% (32/72) and K. pneumonia , 27.8% (20/72). The overall multidrug resistance rate was 86.1% (62/72). ESBL-producers accounted for 41.7% (30/72) of the isolated Enterobacteriaceae . ESBL producing K. pneumonia and E. coli isolates accounted for 70% (14/20) and 37.5% (12/32), respectively. History of UTI in the past 1 year (adjusted odds ratio (AoR) = 0.08, 95%CI (0.01 - 0.57)) and medium family wealth index (AoR = 0.03, 95%CI (0.00 - 0.27)) protected from infection with ESBL-producing Enterobacteriaceae. Conclusion . ESBL production was more common in K. pneumonia and appeared to be a major factor contributing drug resistance UTI in children. The findings call for the need to incorporate ESBL testing in the routine clinical practice. The resistance level to commonly prescribed first-line antibiotics observed within Enterobacteriaceae was alarming calling for strengthened antimicrobial stewardship., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Asnakech Agegnehu et al.)

Objectives: The 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., Methods: 286 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., Results: By 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., Conclusion: AMX/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., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Determination of polymyxins susceptibility by clinical laboratories is a nightmare, mainly because of physicochemical properties of the drug. Elution tests have already been proposed for colistin, but not for polymyxin B. We aimed to evaluate accuracy of Polymyxin B broth disk elution (PBDE) to determine the susceptibility to this drug. We evaluated 196 Enterobacterales (45.9% polymyxin B-resistant). PBDE was done in 15-mL cation-adjusted Mueller-Hinton broth where one polymyxin B disk (300 U) was eluted (2 μg/mL). BMD was performed as reference method. Categorical Agreement (CA), Major Error (ME) and Very Major Error (VME) were 99.5%, 0% and 1.11% (one false-negative K. pneumoniae MIC 4 μg/mL), respectively. As some institutions preferably use polymyxin B over colistin and in some countries colistin are not commercially available, to specifically evaluate polymyxin B is important. PBDE proved to be a cheap and easy to perform methodology to evaluate susceptibility to polymyxin B among Enterobacterales., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: Mueller-Hinton (MH) agar is recommended by EUCAST and CLSI for disc diffusion antimicrobial susceptibility testing. Using EUCAST methodology, we evaluated the performance of 21 internationally available brands of dehydrated MH agar from 17 manufacturers., Methods: MH plates were prepared in-house and evaluated against four quality control (QC) strains tested in triplicate, using EUCAST disc diffusion methodology. This resulted in 30 disc-QC strain combinations and 90 readings per MH brand. All brands were tested blindly and in parallel. Results were evaluated against targets and ranges in the EUCAST QC tables. The agar depth, pH and concentration of five cations were measured for all brands., Results: Six brands of MH agar (Bio-Rad, Biolife, Oxoid, Sigma MH 2, BD BBL MH II and CRITERION) demonstrated excellent performance, with ≥99% of zone diameter readings within QC ranges and ≥70% on target ±1 mm. The poorest performance was seen for Biolab and Merck MH, with 10% (9/90) and 23% (21/90) of readings outside the QC ranges, respectively. Of all readings, 4.9% (93/1890) were out of range, mainly related to trimethoprim sulfamethoxazole (n = 25), aminoglycosides (n = 25) and fluoroquinolones (n = 15). The cation content differed considerably between the agars, and for four brands pH values were outside the acceptable range 7.2-7.4., Discussions: This study evaluated the performance and content of 21 brands of MH dehydrated media. Six brands showed excellent performance with all investigated antimicrobial classes. Others exhibited problems with one or more classes of agents. This could partly be explained by differences in concentration of specific chemical components and pH., (Copyright © 2020 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

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.

The disk diffusion test is very popular but for anaerobes the main pitfalls arise from the significant variation of diameters for an individual MIC and the weak correlation observed between the MIC's values and diameters zone that generates many major and very major errors., Aims of the Study: without any change in the methodology and revisiting only new diameter breakpoints, we try to improve the previous French recommendations and therefore decrease number of errors by introducing recent EUCAST concepts such as ECOFF and ATU Zone., Method: MIC determination by agar dilution was done on 100 anaerobes against 6 antibiotics. Clinical categorization was based on EUCAST Breakpoints. Disk-diffusion method was realized on the same Brucella blood agar incubated in an anaerobic chamber. 550 categorizations by both methods could be done as amoxicillin was not tested on the 50 B. fragilis group. As anaerobic infections are severe and treated by antibiotics at higher dosage, we focus on resistance breakpoint to avoid mainly very major errors (VME). Distribution of inhibition zones for each MIC allow us to fix the zone diameter breakpoints. These results were matched to a large data on distribution of zone diameters for each antibiotic collected from two French hospitals from 1990 to 2005. As example for metronidazole and the B. fragilis group, we calculated the cut-off diameter (15 mm) from a wild type population, at a time when there was no resistance to this antibiotic and observed that it was identical to the diameter breakpoint for susceptibility., Results: For an individual value of MIC, the distribution of diameters is wider for anaerobes especially for clindamycin and metronidazole. Using a 15 mm breakpoint for these two antibiotics limits dramatically the number of very major errors but slowly increases the number of major errors that could be overcome by MIC determination if inhibition zone is less than 15 mm. ATU zones (Area of technical uncertainty) were introduced for amoxicillin-clavulanate (17-20 mm), piperacillin-tazobactam (17-20 mm), imipenem (18-23 mm). Categorization inside the ATU requires MIC determination. Finally, out of 550 determinations, VME were observed in 1.45% of cases, an acceptable rate., Conclusion: in combination with introduction of ATU zones disk diffusion method allows to detect resistant strains with little MIC determinations and very major errors., Competing Interests: Declaration of competing interest The author declares no conflict of interest, monetary or otherwise., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Colistin has become a last-resort antibiotic for the management of multidrug-resistant gram-negative bacteria. The disk diffusion test is cheap and easy to perform but may be unreliable for colistin susceptibility testing due to poor diffusion of the large colistin molecule. An improved agar diffusion test would increase the reliability of colistin susceptibility testing. This study aimed to modify Muller-Hinton agar (MHA) to improve colistin diffusion in agar., Methods: MHA was modified by reducing the agar concentration from 100% to 30% and supplementing with protamine. We tested 60 gram-negative clinical isolates of Pseudomonas aeruginosa (N=27) and Acinetobacter calcoaceticus-baumannii complex (N=33). Disk diffusion test results were interpreted based on minimum inhibitory concentrations determined by broth microdilution., Results: The modified MHA yielded the best performance metrics, including 94.7% sensitivity, 100% specificity, and an area under the curve of 0.995 (95% confidence interval, 0.982-1.000), P <0.001, at a cut-off point of 13 mm., Conclusions: A reduction of the agar concentration from 100% to 30% and the addition of protamine improved colistin diffusion in agar and allowed routine colistin susceptibility testing in a clinical microbiology laboratory, but should be handled with caution., Competing Interests: None., (© The Korean Society for Laboratory Medicine.)

Background: Ceftazidime-avibactam was approved in China in 2019 for treating complicated intra-abdominal infections, hospital-acquired pneumonia, ventilator-associated pneumonia, and infections caused by Enterobacterales and Pseudomonas aeruginosa for which treatment options are limited. However, no currently available commercial systems have been approved for antimicrobial susceptibility testing of ceftazidime-avibactam in China. Here, we evaluated the Etest and disk diffusion method for detecting the activity of ceftazidime-avibactam against Enterobacterales and P. aeruginosa in China., Results: In total, 194 Enterobacterales and 77 P. aeruginosa isolates, which were divided into a random selection group (140 Enterobacterales and 46 P. aeruginosa isolates) and stock group (54 Enterobacterales and 31 P. aeruginosa isolates), were assessed by the Etest, disk diffusion and broth microdilution methods. Minimum inhibitory concentrations and zone diameters were interpreted according to the CLSI supplement M100 30th edition. For all 271 tested isolates, no very major errors were found by using Etest, whereas the overall major error rate was 2.0% (4/203). The overall categorical agreement rates of Etest for Enterobacterales and P. aeruginosa were 99.5% (193/194) and 96.1% (74/77), respectively, and the essential agreement rates were 95.9% (186/194) and 94.8% (73/77), respectively. The disk diffusion method showed that the very major error and major error rates were 1.5% (3/204) and 2.5% (5/203), respectively. Overall categorical agreement rates values of the disk diffusion method for Enterobacterales and P. aeruginosa were 98.5% (191/194) and 93.5% (72/77) compared with broth microdilution, respectively., Conclusions: For Enterobacterales and P. aeruginosa, both the Etest and disk diffusion method showed acceptable performance as alternatives to the standard broth microdilution method for clinical treatment interpretation. Application of the disk diffusion method in Enterobacterales was slightly better than that in P. aeruginosa.

A survey of mycology laboratories for antifungal susceptibility testing (AFST) was undertaken in France in 2018, to better understand the difference in practices between the participating centers and to identify the difficulties they may encounter as well as eventual gaps with published standards and guidelines. The survey captured information from 45 mycology laboratories in France on how they perform AFST (number of strains tested, preferred method, technical and quality aspects, interpretation of the MIC values, reading and interpretation difficulties). Results indicated that 86% of respondents used Etest as AFST method, with a combination of one to seven antifungal agents tested. Most of the participating laboratories used similar technical parameters to perform their AFST method and a large majority used, as recommended, internal and external quality assessments. Almost all the participating mycology laboratories (98%) reported difficulties to interpret the MIC values, especially when no clinical breakpoints are available. The survey highlighted that the current AFST practices in France need homogenization, particularly for MIC reading and interpretation., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Objectives: This study investigated the agreement at the categorical level between the Copan WASPLab incorporating the BioRad expert system against the SIRscan 2000 automatic for antimicrobial disc diffusion susceptibility testing., Methods: The 338 clinical strains (67 Pseudomonas aeruginosa, 19 methicillin-resistant Staphylococcus aureus, 75 methicillin-sensitive S. aureus and 177 Enterobacterales isolates) analysed in this study were non-duplicate isolates obtained from consecutive clinical samples referred to the clinical bacteriology laboratory at Geneva University Hospitals between June and August 2019. For the WASPLab the inoculum suspension was prepared in strict accordance with the manufacturer's instruction (Copan WASP srl, Brescia, Italy) by adding 2 mL of the 0.5 McFarland primary suspension used for the SIRscan analysis into a sterile tube filled with 4 mL of sterile saline (1:3 dilution). The inoculum (2 × 30 μL loop/spreader) was spread over the entire surface of Mueller-Hinton agar plates according to the AST streaking pattern defined by Copan. The antibiotic discs were dispensed by the WASP and inoculated media were loaded on conveyors for transfer to the automatic incubators. The plates were incubated for 16 h, and several digital images were acquired. Inhibition zone diameters were automatically read by the WASPLab and were adjusted manually whenever necessary. For the SIRscan 2000 automatic, the antimicrobial disc diffusion susceptibility testing was performed according to the EUCAST guidelines. The gradient strip method was used to resolve discrepancies., Results: The overall categorical agreement between the compared methods reached 99.1% (797/804; 95% CI 98.2%-99.6%), 99.5% (1029/1034; 95% CI 98.9%-99.8%), and 98.8% (2798/2832; 95% CI 98.3%-99.1%) for P. aeruginosa, S. aureus and the Enterobacterales, respectively., Conclusions: WASPLab incorporating the BioRad expert system provides a fully automated solution for antimicrobial disc diffusion susceptibility testing with equal or better accuracy than other available phenotypic methods., (Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

To comparatively evaluate the performance of the rapid antimicrobial susceptibility testing (AST) system QMAC-dRAST V2.0 and of standard disk diffusion in agar, AST was performed directly from 100 positive blood culture bottles with Gram-negative bacilli. AST results provided by QMAC-dRAST showed 92.9% agreement with disk diffusion method results. Discrepancies observed between results obtained with QMAC-dRAST and disk diffusion method conducted to 10 very major errors (0.8%, S with QMAC-dRAST vs R with disk diffusion method), 40 major errors (3.2%, R vs S, respectively), 15 minor errors (1.2%, S vs I or I vs R, respectively) and 23 very minors errors (1.8%, I vs S or R vs I, respectively). For very major and major errors, in only 36% of the cases did the repeat QMAC-dRAST confirm the initial result, whereas a repeat AST using disk diffusion method confirmed the initial result in 92% of cases. AST results obtained using microdilution in liquid medium confirmed those obtained with QMAC-dRAST and disk diffusion method in 4% and 89%, respectively. Repeatability and reproducibility tests performed on QMAC-dRAST using reference strains showed 94% to 100% of R/S/I categorical agreement., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A three-center study was performed to see if Etest gradient diffusion minimum inhibitory concentration (MIC) methodology correlated with reference broth microdilution (BMD) for antimicrobial susceptibility testing of Burkholderia pseudomallei against six antimicrobial agents known to be usually effective against B. pseudomallei . This study was performed to assist in the decision-making process for possible deployment of the Etest method for antimicrobial susceptibility testing of B. pseudomallei into several regional public health laboratories in the United States. Three laboratories each tested a challenge set of 30 genotypically diverse isolates collected from 15 different countries. MICs were performed by both Etest gradient diffusion and reference BMD for amoxicillin/clavulanate, ceftazidime, doxycycline, imipenem, tetracycline, and trimethoprim/sulfamethoxazole. Etest results for amoxicillin/clavulanate, ceftazidime, doxycycline, and imipenem correlated well with reference BMD by both category interpretation (≥97%) and essential agreement of MIC (≥93%). Tetracycline and trimethoprim/sulfamethoxazole Etests yielded poor correlation with BMD by category interpretation (80%) and essential agreement (70%), respectively. In conclusion, Etest gradient diffusion represents a valid option for antimicrobial susceptibility testing of B. pseudomallei against amoxicillin/clavulanate, ceftazidime, doxycycline, and imipenem. Tetracycline and trimethoprim/sulfamethoxazole Etest results showed some concerning lack of correlation with the corresponding reference BMD results.

The present research was carried out at the Institute of Biotechnology and Genetic Engineering, The University of Agriculture Peshawar, KPK Pakistan. In this study crude methanolic extracts from Thuja occidentalis were tested for their antimicrobial activity against five different bacterial strains (Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Xanthomonas sp. and Staphylococcus aureus) at two different concentrations (10 and 20 mg/ml) using disc diffusion assay. The results showed that Klebsiella pneumoniae was most sensitive to crude extracted sample from leaves at both low and high concentrations measuring 37 and 57% zone of inhibition respectively and Xanthomonas and Staphylococcus aureus was found to be more resistant to the crude extracted samples from leaves at both concentrations. Similarly, Klebsiella pneumoniae was most sensitive to crude extracted samples from seeds at high concentration followed by E. coli at both concentrations. Staphylococcus aureus on the other hand was found to be more resistant to the crude extracted samples at low and high concentrations. These results suggested that extracts prepared from the leaves and seeds of Thuja occidentalis can be used as natural remedy for the treatment of various bacterial infections.

Introduction : The use of antimicrobial discs remains one of the main methods for assessing antibiotic activity. Most discs are made to one of three main standards (FDA, WHO, DIN); these all describe an assay method for assessing the quality of discs using a linear method. Theory predicts a curved relationship, and this is backed up in many cases in practice. In such cases, the assays are potentially invalid. Other sources of uncertainty arising from the manufacturing processes employed are also discussed. Areas covered : This includes error arising from applying FDA, WHO, and DIN standards, the manufacturing techniques employed, and variation in the materials used in production. The need for a specification that relates directly to the use of the discs is also discussed. Expert opinion : Manufactured discs, some of which may be out of specification due to curvature and other sources of error, have been used to establish quality zone sizes. Quality zone sizes have then been used to measure the quality of discs. This circular quality system where there is no quantitative check is potentially unsafe. In the many decades of their use, there has been no comprehensive check on the quality of manufactured discs using quantitative, validated assays.

The disc diffusion test is used for antimicrobial susceptibility testing worldwide. In this study, the performance of both Bio-Rad® antibiotic discs (as compared with Oxoid® discs) and the ADAGIO™ automated system for the reading of disc diffusion test results was evaluated with American Type Culture Collection (ATCC) quality control (QC) and wild strains of bacteria. Inhibition zones of both disc brands were read manually and through use of the ADAGIO™ system. Categorized interpretation of the results for each strain and antibiotic combination was summarized according to the Clinical Laboratory Standards Institute MS-100 (2017 update) manual and ADAGIO™ readings. Eight ATCC QC strains and 120 different wild strains were evaluated, to give a total of 1226 antibiotic/bacteria combinations and 2486 manual readings. One major error and four minor errors (0.08% and 0.34%, respectively) were detected via manual readings of the Bio-Rad® discs as compared with the Oxoid® discs. For the same number of antibiotic/bacteria combinations, five minor errors and one major error (0.42% and 0.08%, respectively) were detected with the Bio-Rad® discs read by the ADAGIO™ system. In addition, the number of times the automatic reading needed manual edition with Bio-Rad® discs was statistically lower than it did with Oxoid® discs (3.7% vs. 5.7%, p < 0.05). These findings support the hypothesis that Bio-Rad discs are not inferior to Oxoid® discs, and the performance of the ADAGIO™ system is comparable to that of manual readings with both disc brands.

Piperacillin-tazobactam (P/T) is a β-lactam-β-lactamase inhibitor combination frequently used in the hospital setting. Etest is a gradient diffusion method that represents an alternative to broth microdilution (BMD) for performing antimicrobial susceptibility testing. We conducted a multicenter evaluation of the performance of the new P/T Etest compared to that of BMD following U.S. Food and Drug Administration (FDA) and International Standards Organization (ISO) standard ISO 20776-2 criteria using Clinical and Laboratory Standards Institute (CLSI)-FDA and European Committee on Antimicrobial Susceptibility Testing (EUCAST) interpretive breakpoints, respectively. A total of 977 isolates (775 Enterobacterales isolates, 119 Pseudomonas aeruginosa isolates, and 83 Acinetobacter baumannii complex isolates) were tested. Overall essential agreement (EA) was 96.4% and 96.6% for Enterobacterales when FDA and ISO 20776-2 criteria, respectively, were followed. EA was 98.3% for P. aeruginosa and 91.6% for the A. baumannii complex when both the FDA and ISO criteria were followed. Applying CLSI-FDA breakpoints, categorical agreement (CA) reached 93.0%, 93.3%, and 89.2% for the Enterobacterales , P. aeruginosa , and the A. baumannii complex, respectively. Two very major errors (VMEs; 1.1%) were found among the Enterobacterales (for 2 Klebsiella pneumoniae isolates). No additional major errors (MEs) or VMEs were found. Applying EUCAST breakpoints, CA was 94.8% and 95.8% for Enterobacterales and P. aeruginosa , respectively (no breakpoints are currently available for the A. baumannii complex). No VMEs were observed among the Enterobacterales , but 2 (0.4%) MEs were found. Among the P. aeruginosa isolates, 2 (6.9%) VMEs and 3 (3.3%) MEs were observed. These errors resulted when P/T Etest MICs were 1 doubling dilution apart from the BMD MICs. In conclusion, the new P/T Etest represents an accurate tool for performing antimicrobial susceptibility testing of Enterobacterales , P. aeruginosa , and A. baumannii complex isolates with limited category errors., (Copyright © 2020 American Society for Microbiology.)

Background: The existing modified carbapenem inactivation methods (mCIMs) recommended by the CLSI for detecting carbapenemase production have not been applicable for Acinetobacter baumannii . We evaluated the influence of matrices used in mCIMs and CIMTris on the stability of the disks for detecting carbapenemase producers and suggested optimal mCIM conditions for detecting carbapenemase-producing A. baumannii ., Methods: Seventy-three A. baumannii isolates characterized for antimicrobial susceptibility and carbapenemase encoding genes were tested for carbapenemase production using mCIM and CIMTris. The influence of the matrices (Tryptic soy broth [TSB] and Tris-HCl) used in these methods on the stability of the meropenem (MEM) disk was also evaluated. The mCIM conditions were adjusted to enhance screening sensitivity and specificity for detecting carbapenemase-producing A. baumannii ., Results: The matrices had an impact on the stability of the MEM disk after the incubation period (two or four hrs). TSB nutrient broth is an appropriate matrix for mCIM compared with Tris-HCl pH 7.6, which leads to the loss of MEM activity in CIMTris. The sensitivity and the specificity of the optimal mCIM were both 100%., Conclusions: We established optimal mCIM conditions for simple, accurate, and reproducible detection of carbapenemase-producing A. baumannii ., Competing Interests: No potential conflicts of interest relevant to this article were reported., (© The Korean Society for Laboratory Medicine.)

Objectives: Until recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommended the cefoxitin disc to screen for mecA-mediated β-lactam resistance in Staphylococcus pseudintermedius. A recent study indicated that cefoxitin was inferior to oxacillin in this respect. We have re-evaluated cefoxitin and oxacillin discs for screening for methicillin resistance in S. pseudintermedius., Methods: We included 224 animal and human S. pseudintermedius isolates from Europe (n = 108) and North America (n = 116), of which 109 were mecA-positive. Disc diffusion was performed per EUCAST recommendations using 30-μg cefoxitin and 1-μg oxacillin discs from three manufacturers and Mueller-Hinton agar from two manufacturers., Results: Cefoxitin inhibition zones ranged from 6 to 33 mm for mecA-positive S. pseudintermedius (MRSP) and from 29 to 41 mm for mecA-negative S. pseudintermedius (MSSP). The corresponding oxacillin zone intervals were 6-20 mm and 19-30 mm. For cefoxitin 16% (95% CI 14.8-18.0%) of the isolates were in the area where positive and negative results overlapped. For oxacillin the corresponding number was 2% (1.6-2.9%). For oxacillin a breakpoint of susceptible (S) ≥ 20 mm and resistant (R) <20 mm resulted in only 0.4% and 1.1% very major error and major error rates respectively., Conclusions: This investigation confirms that the 1-μg oxacillin disc predicts mecA-mediated methicillin resistance in S. pseudintermedius better than the 30-μg cefoxitin disc. For a 1-μg oxacillin disc we propose that 20 mm should be used as cut off for resistance, i.e. isolates with a zone diameter <20 mm are resistant to all β-lactam antibiotics except those with activity against methicillin-resistant staphylococci., (Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Colistin and polymyxin B are old drugs that have been reintroduced to treat Gram-negative infections lacking other treatment options; however, colistin resistance have been reported. To know the correct susceptibility pattern is mandatory for multidrug resistant bacteria. Broth microdilution method is the gold standard to evaluate colistin and polymyxin B susceptibility; nevertheless, it is time consuming and needs expertise to be performed. Disk diffusion method on Müeller-Hinton agar is no longer recommended to evaluate polymyxins susceptibility. In this study we evaluated two methods (disk diffusion in broth and broth macrodilution) as alternative options to identify polymyxin resistance in an easy way. A total of 536 Enterobacteriales isolates were assessed for colistin susceptibility. All non-wild type Enterobacteriales (41) were chosen and 31 wild type bacteria were randomly selected, were used to perform disk diffusion tests in broth and for broth macrodilution tests. We found 100% of concordance between both tested methods and broth microdilution. In conclusion, these two methods are reliable and easier options that complement as initial screening susceptibility for colistin in Enterobacteriales in microbiology laboratories lacking personnel and infrastructure to perform broth microdilution method., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Besides natural and acquired mechanisms of resistance, bacteria can cope with presence of antibiotics by using complex mechanisms such as persistence or tolerance. The main purpose of this study was to evaluate the suitability of newly developed Tolerance Disk Test (TDtest) (Gefen et al., 2017) to detect persistent or tolerant bacterial cells in clinical isolates of Staphylococcus aureus. The principle of the test is to resuscitate the subpopulation of persistent or tolerant bacterial cells following a disk diffusion test by glucose. Results of the TDtest were evaluated using time killing experiments for three pairs of consecutive S. aureus isolates from lower respiratory airway samples of three cystic fibrosis patients with chronic staphylococcal infections. TDtest enabled semi-quantitative detection of persistent or tolerant bacterial populations in all analyzed isolates for oxacillin, vancomycin, and ciprofloxacin to which isolates studied were susceptible. Therefore, TDtest is a promising method for rapidly determining persistence/tolerance in clinical isolates of S. aureus., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A phenotypic assay based on colistin pre-diffusion and differential inhibition of Mobile Colistin Resistance (MCR) protein activity by EDTA showed that, of the 92 strains tested, all MCR producers (49) exhibited an increase ≥5 mm in the inhibition zone around the area of pre-diffusion in the presence of EDTA, in comparison with colistin alone. Results suggest that CPD-E may differentiate MCR-producing microorganisms from resistant microorganisms without this marker., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The optimal method to screen for gastrointestinal colonization with carbapenem-resistant organisms (CRO) has yet to be established. The direct MacConkey (direct MAC) plate method demonstrates high sensitivity for CRO detection, but established zone diameter (ZD) criteria for ertapenem (≤27 mm) and meropenem (≤32 mm) result in high rates of false positives upon confirmatory testing. To increase specificity, we screened for CRO in two high-risk wards using the direct MAC plate method, recorded ZDs for each sample, and generated receiver operating characteristic (ROC) curves to evaluate the optimal ZD cutoff criteria. Of 6,868 swabs obtained over an 18-month period, 4,766 (69%) had growth on MAC plates, and 2,500 (36%) met criteria for further evaluation based on previously established ZDs around the carbapenem disks. A total of 812 (12%) swabs were confirmed positive for at least one CRO and included 213 (3%) carbapenemase-producing organisms (CPO), resulting in a specificity of 78% for the direct MAC plate method. Reducing the ertapenem and meropenem ZDs to ≤25 mm improved specificity to 83%, decreasing the confirmatory testing workload by 32%. The sensitivities with the lower ZD criteria were 89% for CRO and 94% for CPO, respectively. The direct MAC plate method criteria for CRO testing can be modified to balance the sensitivity and specificity of CRO while reducing the burden on clinical microbiology laboratories. These modifications can be particularly helpful in regions with a low CRO prevalence., (Copyright © 2019 American Society for Microbiology.)

Reliable assessment of the susceptibility of animal bacterial pathogens to antimicrobials is of paramount importance in the fight against antimicrobial resistance. This work aims to estimate the repeatability (intra-laboratory agreement) and reproducibility (inter-laboratory agreement) of the disc diffusion assay in veterinary laboratories to understand further if the assay has a role in the surveillance of antimicrobial resistance in animals. Seven major veterinary laboratories from all States in Australia participated, and each tested the same panel of isolates five times at three to four-week intervals, against six antimicrobial agents using Clinical and Laboratory Standards Institute protocols. The panel consisted of twenty different isolates from porcine Escherichia coli from clinical cases and a single reference strain (ATCC 25922). Laboratories were blinded to the identity of the isolates, replicates, and to each other. In total, 4200 inhibition zone diameters (mm) were collected, and analysed descriptively, graphically, and with linear mixed models. Regardless of the laboratories and isolate/antimicrobial combinations, the overall very major error rate (proportion of isolates classified as susceptible when actual status is resistant) was 1.6%; the major error rate (proportion of isolates classified as resistant when actual status is susceptible) was 1.6%; and the 'minor error' rate (proportion of isolates with intermediate susceptibility that measure fully susceptible or resistant or vice versa) was 2.4%. The variation between repeated measurements ranged between 4.4-7.2 mm depending on the antimicrobial agent assessed. The reproducibility was always more variable than the repeatability, which suggested some laboratory effects. The repeatability coefficient of disc diffusion was lowest for tetracycline (4.4 mm, 95% CI: 3.8-5.0 mm) and ampicillin (4.6 mm, 95% CI: 4.2-5.2 mm) and highest for trimethoprim-sulfamethoxazole (6.6 mm, 95% CI: 5.9-7.4 mm). The reproducibility coefficient of disc diffusion was lowest for gentamicin (5.4, 95% CI: 4.0-7.2) and highest for trimethoprim-sulfamethoxazole (7.2 mm, 95%CI: 4.5-11.7 mm). The precision of the disc diffusion assay was deemed satisfactory for use in a national surveillance program for clinical porcine E. coli isolates. However, measurement variation of the disc diffusion assay is of concern for isolates with marginal susceptibility or resistance due to increased risk of misclassification., (Copyright © 2019 Elsevier B.V. All rights reserved.)