EUCAST Research Articles

Yeasts from blood cultures in the wake of the COVID-19 pandemic in a tertiary care hospital: Shift in species epidemiology, steady low antifungal resistance and full in vitro ibrexafungerp activity.

Several institutions reported a rise not only in fungemia incidence but also in the number of cases caused by Candida auris or fluconazole-resistant C. parapsilosis during the COVID-19 pandemic. Since the pandemic broke out in early 2020, we studied its impact on fungemia incidence, species epidemiology, potential patient-to-patient transmission, and antifungal resistance in 166 incident yeast isolates collected from January 2020 to December 2022. Isolates were molecularly identified, and their antifungal susceptibilities to amphotericin B, azoles, micafungin, anidulafungin, and ibrexafungerp were studied following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) method, and genotyped. The fungemia incidence (episodes per 1000 admissions) tended to decrease over time (2020=1.60, 2021=1.36, 2022=1.16); P>.05). Species distribution was C. albicans (50.6%, n=84), C. parapsilosis (18.7%, n=31), C. glabrata (12.0%, n=20), C. tropicalis (11.4%, n=19), C. krusei (3.0%, n=5), other Candida spp. (1.2%, n=2), and non-Candida yeasts (3.0%, n=5). The highest and lowest proportions of C. albicans and C. parapsilosis were detected in 2020. The proportion of isolates between 2020 and 2022 decreased in C. albicans (60.3% vs. 36.7%) and increased in C. parapsilosis (10.3% vs. 28.6%; P<.05) and C. tropicalis (8.8% vs. 16.3%; P>.05). Only three C. albicans intra-ward clusters involving two patients each were detected, and the percentages of patients involved in intra-ward clusters reached 9.8% and 8.0% in 2020 and 2021, respectively, suggesting that clonal spreading was not uncontrolled. Fluconazole resistance (5%) exhibited a decreasing trend (P>.05) over time (2020=7.6%; 2021=4.2%; and 2022=2.1%). Ibrexafungerp showed high in vitro activity.

Laser speckle imaging for visualization of hidden effects for early detection of antibacterial susceptibility in disc diffusion tests.

Rapid identification of effective antibiotic treatment is crucial for increasing patient survival and preventing the formation of new antibiotic-resistant bacteria due to preventative antibiotic use. Currently utilized "gold standard" methods require 16-24 h to determine the most appropriate antibiotic for the patient's treatment. The proposed technique of laser speckle imaging with subpixel correlation analysis allows for identifying dynamics and changes in the zone of inhibition, which are impossible to observe with classical methods. Furthermore, it obtains the resulting zone of inhibition diameter earlier than the disk diffusion method which is recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). These results could improve mathematical models of changes in the diameter of the zone of inhibition around the disc containing the antimicrobial agent, thereby speeding up and facilitating epidemiological analysis.

Novel Antifungals and Aspergillus Section Terrei with Potpourri Susceptibility Profiles to Conventional Antifungals.

The epidemiology of invasive fungal infections (IFIs) is currently changing, driven by aggressive immunosuppressive therapy, leading to an expanded spectrum of patients at risk of IFIs. Aspergillosis is a leading cause of IFIs, which usually affects immunocompromised patients. There are a limited number of antifungal medications available for treating IFIs, and their effectiveness is often hindered by rising resistance rates and practical limitations. Consequently, new antifungals, especially those with novel mechanisms of action, are increasingly required. This study assessed the activity of four novel antifungal agents with different mechanisms of activity, namely, manogepix, rezafungin, ibrexafungerp, and olorofim, against 100 isolates of Aspergillus section Terrei, containing amphotericin-B (AmB)-wildtype/non-wildtype and azole-susceptible/-resistant strains, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) method. In general, all tested agents showed potent and consistent activity against the tested isolates, exhibiting geometric mean (GM) and minimum effective concentration (MEC)/minimum inhibitory concentration (MIC) ranges, respectively, as follows: manogepix (0.048 mg/L, 0.032-0.5 mg/L), rezafungin (0.020 mg/L, 0.016-0.5 mg/L), ibrexafungerp (0.071 mg/L, 0.032-2 mg/L), and olorofim (0.008 mg/L, 0.008-0.032 mg/L). In terms of MIC90/MEC90, olorofim had the lowest values (0.008 mg/L), followed by rezafungin (0.032 mg/L), manogepix (0.125 mg/L), and ibrexafungerp (0.25 mg/L). All the antifungals tested demonstrated promising in vitro activity against Aspergillus section Terrei, including A. terreus as well as azole-resistant and AmB-non-wildtype cryptic species.

In vitro activity of eravacycline and comparators against Gram-negative and Gram-positive bacterial isolates collected from patients globally between 2017 and 2020

To evaluate eravacycline (ERV) activity against Gram-negative and Gram-positive bacteria collected between 2017 and 2020 from worldwide locations. MIC determinations were performed using Clinical and Laboratory Standards Institute (CLSI) broth microdilution methodology. ERV and tigecycline susceptibility was interpreted using United States Food and Drug Administration (FDA) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. Comparator susceptibility was interpreted using CLSI and EUCAST breakpoints. ERV MIC90 was 0.5 μg/mL against 12,436 Enterobacteriaceae isolates, which only increased to 1 μg/mL against multidrug-resistant (MDR) isolates (n=2931 (23.6%). Similar activity was shown against 1,893 Acinetobacter baumannii (MIC90 1 μg/mL) and 356 Stenotrophomonas maltophilia (MIC90 2 μg/mL). ERV was more active against Gram-positive bacteria: 415 Streptococcus pneumoniae (MIC90 0.008 μg/mL), 273 S. anginosus group (MIC90 0.015 μg/mL), 1,876 Enterococcus faecalis and 1,724 E. faecium (MIC90 2 μg/mL), 2,158 Staphylococcus aureus and 575 S. saprophyticus (MIC90 0.12 μg/mL), 1,143 S. epidermidis and 423 S. haemolyticus (MIC90 0.25 μg/mL). ERV MIC90 against methicillin-resistant staphylococci and vancomycin-resistant enterococci was similar to susceptible strains. However, ERV susceptibility varied between EUCAST or FDA against staphylococci, especially S. epidermidis (91.5% vs 47.2%), and vancomycin-resistant E. faecalis (98.3% vs 76.5%). This study reaffirms ERV's consistent broad-spectrum activity that has been evaluated since 2003. ERV remains a key agent for the treatment of bacterial infections, including resistant isolates, but urgent re-assessment of clinical breakpoints is required for staphylococci and enterococci.

Bacterial Skin Microbiota of Seabass from Aegean Fish Farms and Antibiotic Susceptibility of Psychrotrophic Pseudomonas.

Farming seabass (Dicentrarchus labrax) is an essential activity in the Mediterranean basin including the Aegean Sea. The main seabass producer is Turkey accounting for 155,151 tons of production in 2021. In this study, skin swabs of seabass farmed in the Aegean Sea were analysed with regard to the isolation and identification of Pseudomonas. Bacterial microbiota of skin samples (n = 96) from 12 fish farms were investigated using next-generation sequencing (NGS) and metabarcoding analysis. The results demonstrated that Proteobacteria was the dominant bacterial phylum in all samples. At the species level, Pseudomonas lundensis was identified in all samples. Pseudomonas, Shewanella, and Flavobacterium were identified using conventional methods and a total of 46 viable (48% of all NGS+) Pseudomonas were isolated in seabass swab samples. Additionally, antibiotic susceptibility was determined according to standards of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) in psychrotrophic Pseudomonas. Pseudomonas strains were tested for susceptibility to 11 antibiotics (piperacillin-tazobactam, gentamicin, tobramycin, amikacin, doripenem, meropenem, imipenem, levofloxacin, ciprofloxacin, norfloxacin, and tetracycline) from five different groups of antibiotics (penicillins, aminoglycosides, carbapenems, fluoroquinolones, and tetracyclines). The antibiotics chosen were not specifically linked to usage by the aquaculture industry. According to the EUCAST and CLSI, three and two Pseudomonas strains were found to be resistant to doripenem and imipenem (E-test), respectively. All strains were susceptible to piperacillin-tazobactam, amikacin, levofloxacin, and tetracycline. Our data provide insight into different bacteria that are prevalent in the skin microbiota of seabass sampled from the Aegean Sea in Turkey, and into the antibiotic resistance of psychrotrophic Pseudomonas spp.

Rapid antimicrobial susceptibility testing in patients with bacteraemia due to Enterobacterales: an implementation study.

The goal of this descriptive study was to assess the performance as well as the extent of the clinical impact of rapid automated antimicrobial susceptibility testing in patients with bacteraemia due to Enterobacterales. We also aimed to analyse how rapid automated antimicrobial susceptibility testing influences clinical decision-making. This single-centre study conducted at the University Hospital of Zurich included data from all consecutive patients with Enterobacterales bacteraemia from November 2019 to October 2020. There was no control group. The primary outcome was the effect of rapid automated antimicrobial susceptibility testing on antibiotic therapy (no adjustment, escalation to a broader-spectrum antibiotic or de-escalation to a narrower-spectrum antibiotic). Rapid automated antimicrobial susceptibility testing results were further compared to susceptibility tests using European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard methods and erroneous results were noted. Additionally, we investigated turnaround times for rapid automated antimicrobial susceptibility testing and routine diagnostic testing. We analysed 106 patients with 116 episodes of bacteraemia due to Enterobacterales, withEscherichia coliandKlebsiella pneumoniaebeing the most frequent isolates. Almost 8% of pathogens were multidrug resistant. Rapid automated antimicrobial susceptibility testing showed category agreement in 98.4% of all interpretable cases. A significant reduction of more than 20 h in turnaround times could be achieved with rapid automated antimicrobial susceptibility testing compared to the routine diagnostic workflow. In the majority of cases, rapid automated antimicrobial susceptibility testing had no effect, given that the empirical therapy was already correct or circumstances did not allow for de-escalation. In 38.8% of cases, antimicrobial therapy was adjusted, whereas eight cases were de-escalated based on rapid automated antimicrobial susceptibility testing alone. Rapid automated antimicrobial susceptibility testing may be a valuable and safe way to accelerate diagnosis. In particular, time to suitable therapy can be shortened in cases of incorrect therapy. However, physicians are reluctant to de-escalate antibiotic therapy based on rapid automated antimicrobial susceptibility testing alone, limiting its impact in everyday clinics. To further explore the potential of rapid automated antimicrobial susceptibility testing, a stringent/compulsory antibiotic stewardship programme would be a valuable next step.

Amphotericin B, itraconazole, posaconazole, and isavuconazole MICs against clinical Mucorales isolates obtained by visual inspection and spectrophotometric reading according to the EUCAST 9.4 procedure.

We compared the antifungal susceptibility of 92 Mucorales isolates obtained by visual inspection and spectrophotometric readings following EUCAST (European Committee on Antimicrobial Susceptibility Testing) testing. Amphotericin B minimum inhibitory concentrations (MICs) were up to 1 mg/l against most isolates and variable among species, except for Cunninghamella bertholletiae. Posaconazole MICs against most isolates were up to 1 mg/l and high against Mucor circinelloides, some Rhizopus arrhizus, and Rhizopus microsporus. Isavuconazole MICs ranged between 1 and 8 mg/l but were invariably>8 mg/l against M. circinelloides and C. bertholletiae. The agreement between MICs obtained by visual endpoint or spectrophotometric readings was moderate and higher when using the≥90% fungal growth inhibition endpoint.

European Committee on Antimicrobial Susceptibility Testing-Recommended Rapid Antimicrobial Susceptibility Testing of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus From Positive Blood Culture Bottles.

Early diagnosis and treatment are important for a good prognosis of bloodstream infections. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommends rapid antimicrobial susceptibility testing (RAST) based on the disk diffusion methodology for 4, 6, and 8 hours of incubation. We evaluated EUCAST-RAST of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus from positive blood culture bottles. Twenty strains of E. coli, K. pneumoniae, and S. aureus were tested using EUCAST-RAST. Ten antimicrobial agents against E. coli and K. pneumoniae and four agents against S. aureus were tested. The diameter of the inhibition zone (mm) was compared with the minimal inhibitory concentration (μg/mL) obtained using the Sensititre AST system (TREK Diagnostic Systems, East Grinstead, UK). For E. coli, the percentage of total categorical agreement (CA) was 69.5% at 4 hours, and 87% at 8 hours. For K. pneumoniae, the total CA was 89% at 4 hours, and 95.5% at 6 hours. For S. aureus, the total CA was 100% after 4 hours. Discrepancies were observed mainly for E. coli with β-lactam antimicrobial agents, and the numbers of errors decreased over time. EUCAST-RAST for K. pneumoniae and S. aureus met the United States Food and Drug Administration criteria at 6 and 4 hours, respectively, whereas that for E. coli did not meet the criteria for up to 8 hours. RAST can shorten the turn-around testing time by more than one day; therefore, if applied accurately according to laboratory conditions, antimicrobial agent results can be reported faster.

Towards a Standardized Antimicrobial Susceptibility Testing Method for Mycoplasma hyorhinis.

Conducting antimicrobial susceptibility testing (AST) in a comparable manner requires the availability of a standardized method. Organizations, such as the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), provide standardized protocols for a range of fastidious bacteria but not for Mycoplasma hyorhinis. We developed a broth microdilution method for testing M. hyorhinis in a standardized and harmonized way using a modified Friis broth devoid of antimicrobial or otherwise bacterial growth-inhibiting agents. The type strain M. hyorhinis DSM 25591 was chosen to establish the methodology. The antimicrobial agents of interest were doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin, tested by using commercial SensititreTM microtiter plates. In addition, the suitability of the methodology was evaluated via variation of the individual ingredients of the modified Friis broth by either using different batches or choosing other distributors. Despite these alterations, the method provided reliable results. We obtained repeatable minimal inhibitory concentrations for all six tested field isolates and the M. hyorhinis type strain. With this newly proposed method, we aim to provide an improved AST method for diagnostic laboratories and monitoring purposes with better comparability between times and countries. In addition, this new method will allow for an improvement of targeted treatments using antimicrobial agents and thereby reduce the options for resistance development.

Interpretation of Antimicrobial Susceptibility Testing Using European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) Breakpoints: Analysis of Agreement.

Breakpoints provided by European Committee on Antimicrobial Susceptibility Testing (EUCAST) are now being used in many countries. This study was planned to ascertain the agreement in antimicrobial susceptibility using the Clinical and Laboratory Standards Institute (CLSI) and EUCAST breakpoints during the Kirby-Bauer disk diffusion method. This was aprospective observational study. Clinical isolates belonging to the family Enterobacteriaceae recovered between January and December, 2022, were included in the analysis. The diameter of the zone of inhibition of the 14 antimicrobials (viz. amoxicillin/clavulanic acid, cefazolin, ceftriaxone, cefuroxime, cefixime, aztreonam, meropenem, gentamicin, amikacin, ciprofloxacin, levofloxacin, norfloxacin, trimethoprim/sulfamethoxazole and fosfomycin)was analysed. Antimicrobial susceptibility was interpreted using CLSI 2022 and EUCAST 2022 guidelines. Results:Susceptibility data from a total of 356 isolates showed a slight increase in the percentage of resistant isolates with most of the drugs using EUCAST guidelines. The level of agreement varied from almost perfect to slight. For two drugs, i.e., fosfomycin and cefazolin, the agreement was least among the drug analysed (kappa (κ) value < 0.5, p < 0.001). For Ceftriaxone and Aztreonam, with EUCAST, susceptible (S) isolates would have been categorised in the newly redefined "I" category. It would have indicated the use of higher dosages of drugs. Conclusion: Changein the breakpoints impacts the interpretation of the susceptibility. It can also lead to a change in the dosage of the drug used for treatment. Therefore, there is an urgent need to see the impact of recent modifications "I" category of EUCAST on the clinical outcome and usage of antimicrobials.

A HPLC-DAD method to facilitate large-scale therapeutic drug monitoring of dalbavancin

Dalbavancin, a long-acting lipoglycopeptide antibiotic targeting susceptible Gram-positive bacteria, is WHO critically important antibiotic, increasingly used in critical situations such as osteoarticular infections. To ensure its effectiveness and its safety, the therapeutic drug monitoring (TDM) of dalbavancin is strongly recommended. In the absence of an available minimum inhibitory concentration (MIC), the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommends a breakpoint of 0.125 mg/L for Staphylococcus aureus, corresponding to a trough target concentration of 25 mg/L. Nowadays, the TDM is usually performed using a high-performance liquid chromatography (HPLC) method coupled with a tandem mass spectrometry. However, this expensive and specialized equipment and reagents may be difficult to acquire for non-specialized laboratories. The use of HPLC coupled with diode array detector (DAD) facilitates TDM with a lower cost, while preserving the reliability of the results. Our aim was to provide a sensitive and specific method, relying on HPLC-DAD for extending the TDM of dalbavancin beyond non-specialized labs, therefore maximizing its efficiency and Benefit/risk ratio. Our method complied with the European Medicines Agency guidelines of bioanalytical validation. Irrespective of the concentrations of dalbavancin, the coefficient of variation < 10% confirmed the reliability of this analytical method, with a calibration curve ranging from 5 to 100 mg/L. No interferences nor carryover was observed. Our HPLC-DAD method, combined with a simple extraction, provides a widely usable, inexpensive and easy-to-implement new asset for the TDM of Dalbavancin.

Antimicrobial-Resistant Listeria monocytogenes in Ready-to-Eat Foods: Implications for Food Safety and Risk Assessment.

Antimicrobial resistance is an existential threat to the health sector, with far-reaching consequences in managing microbial infections. In this study, one hundred and ninety-four Listeria monocytogenes isolates were profiled for susceptibility using disc diffusion techniques. Possible foodborne listeriosis risk associated with ready-to-eat (RTE) foods (RTEF) and the risk of empirical treatment (EMPT) of L. monocytogenes infections, using multiple antimicrobial resistance indices (MARI) and antimicrobial resistance indices (ARI), respectively, were investigated. Twelve European Committee on Antimicrobial Susceptibility Testing (EUCAST) prescribed/recommended antimicrobials (EPAS) for the treatment of listeriosis and ten non-prescribed antimicrobials (non-PAS)] were evaluated. Antimicrobial resistance > 50% against PAs including sulfamethoxazole (61.86%), trimethoprim (56.19%), amoxicillin (42.27%), penicillin (41.24%), and erythromycin (40.21%) was observed. Resistance > 50% against non-PAS, including oxytetracycline (60.89%), cefotetan (59.28%), ceftriaxone (53.09%), and streptomycin (40.21%) was also observed. About 55.67% and 65.46% of the isolates had MARI scores ranging from 0.25-0.92 and 0.30-0.70 for EPAs and non-PAs, respectively. There was a significant difference (p < 0.01) between the MARI scores of the isolates for EPAs and non-PAs (means of 0.27 ± 0.21 and 0.31 ± 0.14, respectively). MARI/ARI scores above the Krumperman permissible threshold (>0.2) suggested a high risk/level of antimicrobial-resistant L. monocytogenes. The MARI risks of the non-success of empirical treatment (EMPT) attributed to EPAs and non-PAs were generally high (55.67% and 65.463%, respectively) due to the antimicrobial resistance of the isolates. MARI-based estimated success and non-success of EMPT if EUCAST-prescribed antimicrobials were administered for the treatment of listeriosis were 44.329% and 55.67%, respectively. The EMPT if non-prescribed antimicrobials were administered for the treatment of listeriosis was 34.53% and 65.46%, respectively. This indicates a potentially high risk with PAs and non-PAs for the treatment of L. monocytogenes infection. Furthermore, ARI scores ≤ 0.2 for EPAs were observed in polony, potato chips, muffins, and assorted sandwiches, whereas ARI scores for non-PAs were >0.2 across all the RTE food types. The ARI-based estimate identified potential risks associated with some RTE foods, including fried fish, red Vienna sausage, Russian sausage, fruit salad, bread, meat pies, fried chicken, cupcakes, and vetkoek. This investigation identified a high risk of EMPT due to the presence of antimicrobial-resistant L. monocytogenes in RTE foods, which could result in severe health consequences.

A Significant Role of Nontypeable Haemophilus influenzae in Acute Otitis Media in Bulgarian Children

Abstract Objective We conducted a study on Haemophilus influenzae isolates recovered from children with acute otitis media (AOM). We aimed to establish the distribution of noncapsulated (also known as nontypeable Haemophilus influenzae [NTHi]) and encapsulated H. influenzae in the study population, and the antimicrobial susceptibilities of the isolates. Methods We collected 113 nasopharyngeal swabs and 91 middle ear fluids/otorrhea specimens from patients up to 9 years of age with AOM. Of these, 26.1% (n = 53) were culture-positive for H. influenzae. Only one episode of AOM was included per patient. Conventional tests and rapid panel Neisseria/Haemophilus panel were used for the identification of the isolates. Detection of encapsulated and noncapsulated strains was done by polymerase chain reaction (PCR) for bexA gene. PCR-serotyping was performed for capsule types: “a” and “f.” Biotypes were assigned based on the indole, urease, and ornithine decarboxylase activity. Susceptibility testing was performed according to the criteria of European Committee on Antimicrobial Susceptibility Testing (EUCAST). Results Capsule determination showed that 96.2% of H. influenzae isolates responsible for “mild” and “severe” AOM cases in children were NTHi. Biotype I was predominantly associated with AOM isolates. Capsule types “a” and “c” were found in two isolates. Antibiotic resistance was found in 39.6% of the isolates. The highest resistance rate was for trimethoprim-sulfamethoxazole (37.7%). About 20.7% of isolates were ampicillin-resistant: 5.6% expressed a β-lactamase, and 15.1% had a β–lactamase-negative ampicillin-resistant phenotype. Conclusion The current prevalence rates of nonsusceptible H. influenzae to ampicillin appear to be low among AOM. NTHi is an emergent pathogen in AOM cases. Ongoing observations are needed about how NTHi colonizes, survives, and evolves into a leading causative agent of H. influenzae diseases.

Fosfomycin Disk Diffusion Testing among Klebsiella pneumoniae Results in Frequent Inner Colonies and Categorical Disagreement Based on Conflicting Breakpoint Organization Recommendations.

Recent studies indicate that discrete inner colonies (ICs) arise during fosfomycin disk diffusion (DD) testing. CLSI and EUCAST have contradicting recommendations on the interpretation of ICs; CLSI recommends considering them while EUCAST recommends ignoring them when interpreting DD results. We sought to compare the categorical agreement of DD and agar dilution (AD) MIC and to assess the implications of ICs interpretation on zone diameter readings. A convenience sample of 80 Klebsiella pneumoniae clinical isolates with varied phenotypic profiles collected from 3 United States locations was included. Susceptibility was determined in duplicate, using both organization recommendations and interpretations for Enterobacterales. Correlations between methods were calculated using EUCASTIV AD as the reference method. MIC values ranged from 1 to >256 μg/mL with an MIC50/90 of 32/256 μg/mL. Extrapolating EUCASToral and CLSI AD Escherichia coli breakpoints, 12.5% and 83.8% of isolates were susceptible, respectively, whereas 66.3% were susceptible by EUCASTIV AD-which applies to K. pneumoniae. CLSI DD measurements were 2 to 13 mm smaller than EUCAST measurements due to 66 (82.5%) isolates producing discrete ICs. Categorical agreement with EUCASTIV AD was greatest for CLSI AD (65.0%) and poorest for EUCASToral DD (6.3%). Isolates among this collection were frequently classified into different interpretive categories based on varying breakpoint organization recommendations. The more conservative oral breakpoints of EUCAST resulted in more isolates categorized as resistant despite frequent ICs. Differing zone diameter distributions and poor categorical agreement highlight issues of extrapolating E. coli breakpoints and methods to other Enterobacterales, and the clinical relevance of this issue warrants further investigation. IMPORTANCE Fosfomycin susceptibility testing recommendations are complex. Both the Clinical and Laboratory Standards Institute and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recognize agar dilution as the reference method, but they also support disk diffusion as an approved method for Escherichia coli. However, these two organizations have conflicting recommendations for the interpretation of inner colonies that arise during disk diffusion testing which can lead to varying zone diameters and interpretations despite isolates having identical MIC values. Using a collection of 80 Klebsiella pneumoniae isolates, we found that a large (82.5%) portion produced discrete inner colonies during disk diffusion and isolates were frequently classified into different interpretive categories. The more conservative breakpoints of EUCAST resulted in more isolates categorized as resistant despite frequent inner colonies. Differing zone diameter distributions and poor categorical agreement highlight issues of extrapolating E. coli breakpoints and methods to other Enterobacterales, and the clinical relevance of this issue warrants further investigation.

Aspergillus Section Terrei and Antifungals: From Broth to Agar-Based Susceptibility Testing Methods.

Providing timely antifungal treatment to patients suffering from life-threatening invasive fungal infections (IFIs) is essential. Due to the changing epidemiology and the emergence of antifungal resistance in Aspergillus, the most commonly responsible mold of IFIs, antifungal susceptibility testing (AFST) has become increasingly important to guide clinical decisions. This study assessed the essential agreement (EA) between broth microdilution methods (the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST)) and the Etest of amphotericin B (AmB), liposomal amphotericin B (L-AmB), and isavuconazole (ISA) against 112 Aspergillus section Terrei. An EA within ±2 dilutions of ≥90% between the two methods was considered acceptable. Excellent EA was found between EUCAST and CLSI of AmB and ISA (98.2% and 95.5%, respectively). The correlation of Etest results and EUCAST/CLSI was not acceptable (<90%) for any tested antifungal; however, Etest and CLSI for AmB (79.6%) and ISA (77.6%) showed a higher EA than Etest and EUCAST for AmB (49.5%) and ISA (46.4%). It was concluded that the Etest method requires its own clinical breakpoints (CBPs) and epidemiological cutoff values (ECVs), and interpreting Etest results using EUCAST and CLSI-adapted CBPs and ECVs could result in misinterpretation as Etest shows lower minimum inhibitory concentrations (MICs).

Genetic determinants of macrolide and tetracycline resistance in penicillin non-susceptible Streptococcus pneumoniae isolates from people living with HIV in Dar es Salaam, Tanzania

BackgroundOver one million yearly deaths are attributable to Streptococcus pneumoniae and people living with HIV are particularly vulnerable. Emerging penicillin non-susceptible Streptococcus pneumoniae (PNSP) challenges therapy of pneumococcal disease. The aim of this study was to determine the mechanisms of antibiotic resistance among PNSP isolates by next generation sequencing.MethodsWe assessed 26 PNSP isolates obtained from the nasopharynx from 537 healthy human immunodeficiency virus (HIV) infected adults in Dar es Salaam, Tanzania, participating in the randomized clinical trial CoTrimResist (ClinicalTrials.gov identifier: NCT03087890, registered on 23rd March, 2017). Next generation whole genome sequencing on the Illumina platform was used to identify mechanisms of resistance to antibiotics among PNSP.ResultsFifty percent (13/26) of PNSP were resistant to erythromycin, of these 54% (7/13) and 46% (6/13) had MLSB phenotype and M phenotype respectively. All erythromycin resistant PNSP carried macrolide resistance genes; six isolates had mef(A)-msr(D), five isolates had both erm(B) and mef(A)-msr(D) while two isolates carried erm(B) alone. Isolates harboring the erm(B) gene had increased MIC (> 256 µg/mL) towards macrolides, compared to isolates without erm(B) gene (MIC 4-12 µg/mL) p < 0.001. Using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, the prevalence of azithromycin resistance was overestimated compared to genetic correlates. Tetracycline resistance was detected in 13/26 (50%) of PNSP and all the 13 isolates harbored the tet(M) gene. All isolates carrying the tet(M) gene and 11/13 isolates with macrolide resistance genes were associated with the mobile genetic element Tn6009 transposon family. Of 26 PNSP isolates, serotype 3 was the most common (6/26), and sequence type ST271 accounted for 15% (4/26). Serotypes 3 and 19 displayed high-level macrolide resistance and frequently carried both macrolide and tetracycline resistance genes.ConclusionThe erm(B) and mef(A)-msr(D) were common genes conferring resistance to MLSB in PNSP. Resistance to tetracycline was conferred by the tet(M) gene. Resistance genes were associated with the Tn6009 transposon.

Evaluation of Variability in Interpretation of Disk Diffusion Testing for Cefiderocol Using Different Brands of Mueller-Hinton Agar.

Cefiderocol is a new antibiotic used to treat infections with antibiotic resistant Gram-negative bacilli. The impact of differences between Mueller-Hinton agar (MHA) brands on susceptibility testing is underexplored. Compounding the implementation of cefiderocol susceptibility testing is a lack of harmonization between different regulatory body breakpoint criteria. We performed Kirby-Bauer disk diffusion using BD, Hardy, and Remel MHA, in addition to broth microdilution for Acinetobacter baumannii (n = 25), Enterobacterales (n = 25), Stenotrophomonas maltophilia (n = 24), and Pseudomonas aeruginosa (n = 23). We analyzed disk diffusion diameters and minimum inhibitory concentrations using interpretive criteria from the Clinical and Laboratory Standards Institute (CLSI), US Food and Drug Administration (FDA), and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint criteria impacted interpretation of susceptibly testing results, for example with the broth microdilution we found 8% (2/25) of A. baumannii isolates change interpretation between CLSI and EUCAST and 32% (8/25) change between CLSI and FDA, 12% (3/25) of Enterobacterales change between CLSI and EUCAST, 13% (3/23) of P. aeruginosa interpretations change between CLSI and FDA, and 4% (1/25) S. maltophilia change between CLSI and FDA. There was a significant difference between the zone disk diffusion diameters for P. aeruginosa and S. maltophilia between Hardy and BD; which changed interpretation (using CLSI criteria) for 8.7% (2/23) for P. aeruginosa but 0% (0/24) for S. maltophilia. Breakpoint criteria impact cefiderocol susceptibility testing interpretation for broth microdilution and disk diffusion. Choice of MHA brand can also affect result interpretation.

The European committee on antimicrobial susceptibility testing disc diffusion susceptibility testing method for frequently isolated anaerobic bacteria

ObjectivesAntimicrobial resistance in anaerobic bacteria is increasing and there is a link between inappropriate antimicrobial therapy and poor clinical outcome in the treatment of infections caused by anaerobic bacteria. Accurate and timely antimicrobial susceptibility testing of anaerobic bacteria is therefore of critical importance. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) has recently described a disc diffusion susceptibility testing method for anaerobic bacteria using fastidious anaerobe agar (FAA) supplemented with 5% defibrinated horse blood (HB). This method was previously validated for Bacteroides spp. only. The aim of this study was to determine the suitability of FAA-HB for disc diffusion and also for frequently isolated anaerobic bacteria. MethodsClinical isolates, including 54 Bacteroides/Phocaeicola/Parabacteroides spp., 49 Prevotella spp., 51 Fusobacterium necrophorum, 58 Clostridium perfringens, and 54 Cutibacterium acnes were evaluated against six antimicrobial agents. MICs were determined by agar dilution following Clinical and Laboratory Standards Institute methodology, modified to use FAA-HB as recommended by EUCAST, instead of supplemented Brucella agar, and disc diffusion was performed on FAA-HB following EUCAST methodology. ResultsResults for quality control strains were reproducible, with 99.3% of zones within range. Disc diffusion by EUCAST methodology was able to distinguish between susceptible and resistant isolates of anaerobic bacteria for benzylpenicillin, piperacillin-tazobactam, meropenem, clindamycin, and metronidazole (98.7% correct categorization). No isolates resistant to vancomycin were tested, but zone diameters correctly categorized the susceptible isolates, and there was a logical relationship between MICs and inhibition zones. DiscussionThe recently published EUCAST method for disc diffusion for anaerobic bacteria based on FAA-HB is a reproducible and accurate method for susceptibility testing of frequently isolated anaerobic bacteria.

Evaluation of Pathogens and Antibiotic Resistance Profiles Isolated from Lower Respiratory Tract Samples

Objective: In this study, we aimed to guide clinicians in planning empirical treatment and contribute to regional data by presenting bacterial lower respiratory tract infection agents and antibiotic resistance profiles. Methods: All lower respiratory tract samples from patients admitted to our laboratory between January 01 and December 31, 2021 were evaluated retrospectively. Bacterial identification and susceptibility tests were performed using the VITEK 2 automated system and evaluated in accordance with the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Results: Significant growth was detected in 461 of 923 lower respiratory tract samples. Antibiotic susceptibility test results of 340 samples were evaluated after excluding the repeated samples of same patient. Gram negative bacteria was isolated in 309 (90.9%), Staphylococcus aureus in 17 (5%) and Candida albicans in 14 (4.1%) of 340 samples. The most frequently isolated Gram-negative bacteria were Pseudomonas aeruginosa (121, 39.2%), Acinetobacter baumannii (88, 28.5%), Klebsiella pneumoniae (64, 20.7%) and Escherichia coli (36, 11.6%), respectively. Resistance rates of A. baumannii isolates to carbapenems, piperacillin/tazobactam, ceftazidime and fluoroquinolones were found to be over 90%, and higher resistance was observed in intensive care units (ICU) than in wards. Conclusion: The increased antibiotic resistance observed in lower respiratory tract infections from hospitalized patients in ICUs, is remarkable. As a result, knowing the causative agents and current resistance profiles is important, especially for hospitalized patients in ICUs, in order to initiate appropriate empirical treatment and to ensure treatment success. The data obtained from our study will guide clinicians in planning empirical treatment.

Ciprofloxacin resistance and tolerance of Pseudomonas aeruginosa ocular isolates

PurposeTolerance to antibiotics may occur due to changes in bacterial growth patterns and can be a precursor to development of resistance. However, there is a lack of information on the ability of ocular bacteria isolates to develop tolerance. This paper explores the tolerance to 8 different antibiotics of 61 microbial keratitis isolates of Pseudomonas aeruginosa from Australia and India using the MBC/MIC ratio, with tolerance defined by a ratio ≥ 32, and tolerance to ciprofloxacin by an agar diffusion assay. MethodsAntibiotics used were ciprofloxacin, levofloxacin, gentamicin, tobramycin, piperacillin, imipenem, ceftazidime and polymyxin B. Isolates were sourced from microbial keratitis infections in Australia and India. Minimum bactericidal and minimum inhibitory concentration (MBC and MIC) were obtained using broth microdilution and compared to breakpoints from the Clinical Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) to determine bacterial susceptibility. Tolerance was assessed as MBC/MIC ≥ 32. An alternative method for tolerance detection (TD) was assessed with 13P. aeruginosa sensitive isolates by agar disk diffusion assay of ciprofloxacin followed by application of glucose to the agar and observation of re-growth of colonies. ResultsThirty-three isolates were resistant to imipenem, 20 to ciprofloxacin, 14 to tobramycin and piperacillin, 12 to levofloxacin and ceftazidime, 8 to gentamicin, and 5 to polymyxin B. The percentage of strains resistant to levofloxacin (7 vs 30 %; p = 0.023), gentamicin (0 vs 24 %; p = 0.005) and tobramycin (4 vs 33 %; p = 0.004) was significantly greater in isolates from India.On average, strains from India exhibited notably greater MIC and MBC values compared to strains obtained from Australia. Out of 61 isolates, none displayed an MBC/MIC ratio ≥ 32. However, three sensitive isolates had low tolerance, nine had medium tolerance and one had high tolerance to ciprofloxacin with the TDtest. ConclusionsThis study used two methods to determine whether P. aeruginosa strains could show tolerance to antibiotics. Using the MBC/MIC criteria no strain was considered tolerant to any of the eight antibiotics used. When 13 strains were tested for tolerance against ciprofloxacin, the most commonly used monotherapy for keratitis, one had high tolerance and nine had medium tolerance. This demonstrates the capacity of P. aeruginosa to develop tolerance which may result in therapeutic failures if inappropriate dosing regimens are used to treat keratitis.