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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.

Background: Coffee is a major dietary source of polyphenols. Previous research found that coffee had a protective effect on periodontal disease. In this study, we aimed to investigate whether coffee extract and its primary phenolic acid, chlorogenic acid, affect the growth and protease activity of a periodontopathogen Porphyromonas gingivalis (P. gingivalis). Methods: Coffee extract and chlorogenic acid were prepared by a two-fold serial dilution. The turbid metric test and plate count method were used to examine the inhibitory effects of chlorogenic acid on P. gingivalis. The time-kill assay was used to measure changes in the viability of P. gingivalis after exposure to chlorogenic acid for 0–24 h. The protease activity of P. gingivalis was analyzed using the optical density of a chromogenic substrate. Results: As a result, the minimum inhibitory concentration (MIC) of chlorogenic acid was 4 mg/mL, and the minimum bactericidal concentration was 16 mg/mL. Chlorogenic acid at concentrations above MIC resulted in a longer-lasting inhibitory effect on P. gingivalis viability and significantly reduced associated protease activity. The coffee extract showed antibacterial activity as observed by the disk diffusion test, whereas these inhibitory effects were not affected by different roast degrees of coffee. Conclusions: Collectively, our novel findings indicate that chlorogenic acid not only has antimicrobial activity but also reduced the protease activity of P. gingivalis. In addition, coffee extract inhibits the proliferation of P. gingivalis, which may partly be attributed to the effect of chlorogenic acid.

Introduction: Antimicrobial resistance (AMR) is a global health challenge, and antimicrobial susceptibility testing (AST) is vital for guiding treatment. Although widely used, the Kirby-Bauer method depends on skilled interpretation, which can be time-intensive and error-prone. This study explored the potential of an artificial intelligence (AI)-driven progressive web app (PWA) to automate the analysis of Kirby-Bauer test images, thereby enhancing accuracy and efficiency. Methodology: Images of Kirby-Bauer test results were annotated to train the Faster R-CNN ResNet-50 to detect agar plates, inhibition zones, and antibiotic discs. MobileNetv2 was used for antibiotic disc classification. A Human-in-the-Loop (HITL) approach enabled technicians to correct errors and improve model performance through retraining. The PWA, built with VueJS and Python-PHP, provided real-time analysis aligned with the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards. Results: The application achieved 92.95% accuracy for inhibition zone detection and 96.92% accuracy for antibiotic disc identification, with a performance improvement of 99.28% following HITL corrections. The measurements closely aligned with those of the technicians in 89.54% of the cases. The system processed up to 50 images per hour, supporting reliable and rapid AST workflow. Conclusions: The AI-powered “Disc Diffusion Reader” demonstrated high accuracy and efficiency, by reducing interpretation variability in the AST workflows. Its scalability and adaptability, particularly in low-resource settings, make it a valuable tool for combating AMR. Continuous retraining and validation will ensure sustained reliability, and highlight the potential of AI-driven solutions in modern microbiology.

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.

Background Drug susceptibility testing (DST) for Nocardia spp. is essential to initiate effective antibiotic therapy. Currently, the only recommended technique is the determination of minimum inhibitory concentrations (MICs) by microdilution. This method can be tedious to perform, despite the availability of ready-to-use plates. Herein, the aim was to determine the critical inhibition diameters specific to Nocardia spp. Methods MICs of 134 Nocardia isolates were determined by microdilution. Interpretative categories (Susceptible/Intermediate/Resistant) were determined using Clinical and Laboratory Standards Institute breakpoints. In parallel, disk diffusion DST was performed. Receiver-operating-characteristic (ROC) curves were constructed to determine the inhibition diameter value that best discriminated between susceptible and non-susceptible strains (intermediate/resistant). The category agreement (CA), the rate of major (maj) and very major (vmj) discrepancies between microdilution and disk diffusion method was calculated. Results For tobramycin, the critical diameter of 19 mm (diameter ≤ 19 mm = resistant strain; diameter > 19 mm = susceptible strain) provided a CA of 98.5%, 0.0% vmj, and 2.9% maj discrepancies, reaching strictly the acceptable performance criteria defined by the U.S. Food and Drug Administration (FDA). For amikacin, the critical diameter of 25 mm (diameter ≤ 25 mm = resistant strain; diameter > 25 mm = susceptible strain) provided a CA of 98.5%, 0.0% vmj, and 1.5% maj discrepancies. For imipenem, excluding N. farcinica and N. cyriacigeorgica , the critical diameter of 29 mm (diameter ≤ 29 mm = resistant strain; diameter > 29 mm = susceptible strain), provided a CA of 98.6%, 0.0% vmj, and 0.0% maj discrepancies. Despite an estimated vmj rate 0.0%, the 95%-confident-interval exceeded the FDA criteria due to an insufficient number of amikacin/imipenem-resistant strains. For other tested antibiotics (ciprofloxacin, moxifloxacin, amoxicillin-clavulanate, ceftriaxone, cotrimoxazole, linezolid), the FDA criteria were not reached. Conclusions Although the FDA criteria were mostly unmet, disk diffusion DST was suitable to accurately categorize Nocardia isolates into interpretative categories for the aminoglycosides and imipenem only, excluding species N. farcinica and N. cyriacigeorgica .

The objectives of this study were to define the shortest incubation times on the WASPLab for reliable MALDI-TOF/MS-based species identification and for the preparation of a 0.5 McFarland suspension for antimicrobial disk diffusion susceptibility testing using short subcultures growing on solid culture media inoculated by positive blood cultures spiked with a wide range of pathogens associated with bloodstream infections. The 520 clinical strains (20 × 26 different species) included in this study were obtained from a collection of non-consecutive and non-duplicate pathogens identified at Geneva University Hospitals. After 4 h of incubation on the WASPLab, microorganisms’ growth allowed accurate identification of 73% (380/520) (95% CI, 69.1–76.7%) of the strains included in this study. The identification rate increased to 85% (440/520) (95% CI, 81.3–87.5%) after 6-h incubation. When excluding Corynebacterium and Candida spp., the microbial growth was sufficient to permit accurate identification of all tested species (100%, 460/460) (95% CI, 99.2–100%) after 8-h incubation. With the exception of Burkholderia cepacia and Haemophilus influenzae, AST by disk diffusion could be performed for Enterobacterales and non-fermenting Gram-negative bacilli after only 4 h of growth in the WASPLab. The preparation of a 0.5 McFarland suspension for Gram-positive bacteria required incubation times ranging between 3 and 8 h according to the bacterial species. Only Corynebacterium spp. required incubation times as long as 16 h. The WASPLab enables rapid pathogen identification as well as swift comprehensive AST from positive blood cultures that can be implemented without additional costs nor hands-on time by defining optimal time points for image acquisition.

Sea cucumbers are marine organisms with uses in food, cosmetics, and medicine. This study aimed to identify Indonesian sea cucumbers with high antioxidant and antibacterial activities. Twenty-one sea cucumber species were used for this study. Antioxidant capacity was evaluated using the 2,2-diphenyl-β-picrylhydrazine assay. Antibacterial activity was assessed using the disk diffusion assay, whereas the resazurin-based assay was employed to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Volatile compounds possibly related to the biological activity of sea cucumbers were analyzed via gas chromatography–mass spectrometry (GC–MS). Holothuria atra had the strongest antioxidant capacity (IC50 = 14.22 ± 0.87 µg µL−1). Stichopus vastus displayed the best antibacterial activity against Staphylococcus aureus, whereas Stichopus ocellatus extract was most potent against Vibrio cholerae. Holothuria albiventer, which controlled Bacillus subtilis most effectively while also being active against S. aureus and V. cholerae, was the optimal antimicrobial species. H. albiventer and Actinopyga echinites inhibited B. subtilis growth at 12.5 µg µL−1. The MBC tests indicated that the antibacterial activities of sea cucumbers at the MIC were bacteriostatic, rather than bactericidal, in nature. GC–MS analysis uncovered long-chain fatty acids that might be associated with the antibacterial activities of sea cucumbers.

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.

In our tertiary care center, the reported susceptibility of E. coli blood isolates to amoxicillin/clavulanic acid exceeded 90% in 2005 and showed a progressive decrease to 50% by 2017. In this study, we investigate whether there is a real increase in resistant E. coli strains or if this apparent decline in reported susceptibility might be attributed to the substitution of CLSI by EUCAST guidelines in 2014. We randomly selected 237 E. coli blood isolates (stored at − 80 °C) from 1985 to 2018 and reassessed their MIC values, applying both the CLSI (fixed ratio of clavulanic acid) and EUCAST guidelines (fixed concentration of clavulanic acid). In parallel, the susceptibility of these isolates was retested by disk diffusion, according to the EUCAST guidelines. Whole genome sequencing was successfully performed on 233 of the 237 isolates. In only 130 of the 237 isolates (55.0%), testing according to the EUCAST and CLSI criteria delivered identical MIC values for amoxicillin/clavulanic acid. In 64 of the 237 isolates (27.0%), the MIC values diverged one dilution; in 38 (16.0%), two dilutions; and in five (2.1%), three dilutions. From these 107 discrepant results, testing according to EUCAST methodology revealed more resistant profiles in 93 E. coli strains (94.1%). Also, phenotypical susceptibility testing according to EUCAST guidelines tends to correlate better with the presence of beta-lactamase genes compared to CLSI testing procedure. This study highlights the low agreement between EUCAST and CLSI methodologies when performing MIC testing of amoxicillin/clavulanic acid. More strains are categorized as resistant when EUCAST guidelines are applied. The low agreement between EUCAST and CLSI was confirmed by WGS, since most of EUCAST resistant/CLSI sensitive isolates harbored beta-lactamase genes.

The spread of antimicrobial resistance is one of the major health emergencies of recent decades. Antimicrobial-resistant bacteria threaten not only humans but also populations of domestic and wild animals. The purpose of this study was to evaluate the distribution of antibiotic resistance (AMR) and multidrug resistance (MDR) in bacterial strains isolated from six Southern-Italian bat populations. Using the disk diffusion method, we evaluated the antimicrobial susceptibility of 413 strains of Gram-negative bacteria and 183 strains of Gram-positive bacteria isolated from rectal (R), oral (O) and conjunctival (C) swabs of 189 bats belonging to 4 insectivorous species (Myotis capaccinii, Myotis myotis, Miniopterus schreibersii and Rhinolophus hipposideros). In all bat species and locations, numerous bacterial strains showed high AMR levels for some of the molecules tested. In both Gram-negative and Gram-positive strains, the resistance patterns ranged from one to thirteen. MDR patterns varied significantly across sites, with Grotta dei Pipistrelli in Pantalica displaying the highest levels of MDR (77.2% of isolates). No significant differences were found across different bat species. Monitoring antibiotic resistance in wildlife is a useful method of evaluating the impact of anthropic pressure and environmental pollution. Our analysis reveals that anthropic contamination may have contributed to the spread of the antibiotic resistance phenomenon among the subjects we examined.