Development of Rapid Disk Diffusion Device Using Laser Speckle Formation Technology for Rapid Antimicrobial Susceptibility Testing.

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.

Maximally stable extremal regions-based algorithm for automatic interpretation of disc-diffusion antibiotic sensitivity test.

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.

Evaluation of Automated Disk Diffusion Antimicrobial Susceptibility Testing Using Radian® In-Line Carousel.

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.

Comparison of BD Phoenix and disk diffusion to broth microdilution for determining cefepime susceptibility among carbapenem-resistant Enterobacterales.

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 (CPblaKPC-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 CPblaKPC-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 CPblaKPC-CRE and non-CP CRE with BD Phoenix and DD tests. For error rate-bounded method, miEs were <40% for IHigh + 1 to ILow - 1 ranges for CPblaKPC-CRE and non-CP CRE with BD Phoenix. Regarding disk diffusion, miEs were unacceptable for all MIC ranges among CPblaKPC-CRE. For non-CP CRE isolates, only IHigh + 1 to ILow - 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 CPblaKPC-CRE than non-CP CRE.

Multicenter comparison of Etest, Vitek2 and BD Phoenix to broth microdilution for beta-lactam susceptibility testing of Streptococcus pneumonia.

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.

Possible impact of revisions in disc diffusion breakpoints for aminoglycosides and piperacillin/tazobactam in the 33rd edition of CLSI M100 document on clinical reporting and use in Indian settings with low susceptibility.

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.

Performance of disk diffusion method for aztreonam in combination with avibactam against Enterobacteriales.

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.

Ampicillin susceptibility testing of Haemophilus influenzae in the routine clinical laboratory by the EUCAST methodology compared to broth microdilution and the presence of ftsI gene mutations.

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 ß-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.

Características clínicas y microbiológicas de la infección de vías urinarias bajas en población ambulatoria / Clinical and Microbiological Characteristics of Lower Urinary Tract Infections in an Outpatient Population

Introducción: El tratamiento de las infecciones del tracto urinario es casi siempre empírico, lo que genera una serie de problemas en la consulta diaria. Objetivo: Caracterizar clínica y microbiológicamente las infecciones de vías urinarias bajas no complicadas en pacientes de una clínica de primer nivel. Métodos: Se realizó un estudio transversal descriptivo. La identificación de las bacterias del cultivo de orina se efectuó por métodos establecidos. La prueba de susceptibilidad a los antimicrobianos se realizó por la técnica Kirby-Bauer. Se utilizó el programa estadístico SPSS versión 26, con la prueba de ji al cuadrado y un análisis multivariado discriminante. Se calculó también razón de momios con el programa Epi-Info. Resultados: Se incluyeron 270 pacientes, con frecuencia de 39,3 por ciento de cultivos positivos, y Escherichia coli como la especie predominante. Se identificaron, además, 31,3 por ciento de bacterias Gram positivas. Se presentó significancia estadística entre la infección urinaria y factores como el sexo, y la infección del tracto urinario previa en las mujeres. Se obtuvo 100 por ciento de cepas resistentes a ampicilina. En general, se obtuvieron porcentajes de resistencia altos en los antimicrobianos probados. Conclusiones: Escherichia coli fue la especie más frecuentemente aislada, sin embargo, existe una serie de microorganismos implicados en enfermedades del tracto genital como Gardnerella vaginalis, que parecen estar involucrados en la etiología de las infecciones del tracto urinario. Se identificaron factores de riesgo como el sexo biológico y las infecciones previas en mujeres. Se obtuvieron porcentajes de resistencia altos en los antimicrobianos probados(AU)

Introduction: The management of urinary tract infections is almost always empirical, which generates a series of problems in the daily consultation. Objective: To characterize, clinically and microbiologically, uncomplicated lower urinary tract infections in patients of a primary level clinic. Methods: A descriptive and cross-sectional study was carried out. Bacterial identification in urine culture was performed by established methods. Antimicrobial susceptibility testing was performed using the Kirby-Bauer technique. The statistical software SPSS (version 26) was used, with the chi squared test and multivariate discriminant analysis. Odds ratios were also calculated with the Epi-Info program. Results: A total of 270 patients were included, with a 39.3percent frequency of positive cultures and Escherichia coli as the predominant species. In addition, 31.3percent of Gram-positive bacteria were identified. There was statistical significance between urinary tract infection and factors such as sex or previous urinary tract infection in women. One result was 100percent of ampicillin-resistant strains. In general, high percentages of resistance were obtained for the tested antimicrobials. Conclusions: Escherichia coli was the most frequently isolated species; however, there is a number of microorganisms implicated in genital tract diseases, such as Gardnerella vaginalis, which appear to be involved in the etiology of urinary tract infections. Risk factors such as biological sex and previous infections in women were identified. High percentages of resistance were obtained for the tested antimicrobials(AU)

Comparison of carbapenem minimum inhibitory concentrations of Oxacillin-48-like Klebsiella pneumoniae by Sensititre, Vitek 2, MicroScan, and Etest.

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.

Multicenter Evaluation of a Gradient Diffusion Method for Antimicrobial Susceptibility Testing of Helicobacter pylori.

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.

Rapid determination of ceftazidime/avibactam susceptibility of carbapenemase-producing Enterobacterales directly from blood cultures: a comparative evaluation of EUCAST disc diffusion RAST and direct Etest® RAST.

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.

Stop Waiting for Tomorrow: Disk Diffusion Performed on Early Growth Is an Accurate Method for Antimicrobial Susceptibility Testing with Reduced Turnaround Time.

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 (r2 = 0.98) and 10 h (r2 = 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.

Assessment of Ceftazidime-Avibactam 30/20-µg Disk, Etest versus Broth Microdilution Results When Tested against Enterobacterales Clinical Isolates.

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 ß-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 ß-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.

Impact of the introduction of EUCAST's concept of "area of technical uncertainty".

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.

Multicenter Clinical Evaluation of ETEST Plazomicin (PLZ) for Susceptibility Testing of Enterobacterales.

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.

Machine learning approach for predicting the antifungal effect of gilaburu (Viburnum opulus) fruit extracts on Fusarium spp. isolated from diseased potato tubers.

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.

Novel Disk Diffusion Assay on Magnesium Oxalate Agar To Evaluate the Susceptibility of Yersinia pestis to Type III Secretion System Inhibitors.

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 (dH2O) 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.

Culture-Based Antimicrobial Susceptibility Testing for Helicobacter pylori.

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.