Reference Broth Microdilution Research Articles

Assessing the Efficacy of Chlorhexidine in Combating Most Important Causative Agents of Fungal Keratitis: An in Vitro Comparative Study With Seven Antifungal Agents

Purpose Fungal keratitis (FK) is a difficult condition to treat, particularly in underdeveloped nations. The study aimed to compare the in vitro activity of chlorhexidine (CHX) and seven antifungal agents against a collection of fungi recovered from FK patients. Methods Seventy-three fungi were collected from patients with FK included in study. The antifungal agents tested were fluconazole, voriconazole, posaconazole, miconazole, natamycin, amphotericin B, and caspofungin. The concentration range for CHX was 1–1024 μg/mL. Assessments of antifungal susceptibility were conducted using the EUCAST broth microdilution reference method. Results The findings demonstrated the beneficial in vitro inhibition of filamentous and yeast fungi by CHX. CHX demonstrated efficacy against Fusarium spp., Aspergillus spp., Candida spp., S. apiospermum and dematiceous fungi at concentrations of 4–64, 32–64, 4–32, 8, and 4–16 µg/mL respectively. The median MICs and MIC distributions of CHX showed no significant differences among the evaluated spp. (p > 0.05). The most effective antifungal drug was posaconazole, which was followed by voriconazole, caspofungin, and amphotericin B. Conclusion In situations where access to a range of antifungal medications and microbiological facilities is limited, CHX should be further investigated as a potential treatment for FK. It might be able to treat the condition as an inexpensive topical treatment.

Assessment of the activity and mechanisms of resistance to cefiderocol and combinations of β-lactams and the novel β-lactamase inhibitors avibactam, taniborbactam, zidebactam, nacubactam, xeruborbactam, and ANT3310 in emerging double-carbapenemase-producing Enterobacterales.

We aimed to investigate the activity of and mechanisms of resistance to cefiderocol and innovative β-lactam/β-lactamase inhibitor combinations in a nationwide collection of double-carbapenemase-producing Enterobacterales. In all, 57 clinical isolates co-producing two carbapenemases collected from Spanish hospitals during the period 2017-2022 were analyzed. Minimum inhibitory concentration (MIC) values for ceftazidime, ceftazidime/avibactam, aztreonam, aztreonam/avibactam, aztreonam/nacubactam, cefiderocol, cefepime, cefepime/taniborbactam, cefepime/zidebactam, cefepime/nacubactam, imipenem, imipenem/relebactam, meropenem, meropenem/vaborbactam, meropenem/xeruborbactam, and meropenem/ANT3310 were determined by reference broth microdilution. Genetic drivers of resistance were analyzed by whole-genome sequencing (WGS). The collection covered nine carbapenemase associations: VIM + OXA-48 (21/57), NDM + OXA-48 (11/57), KPC + VIM (10/57), KPC + OXA-48 (6/57), IMP + OXA-48 (3/57), NDM + KPC (2/57), NDM + VIM (2/57), NDM + GES (1/57), and KPC + IMP (1/57). Ceftazidime/avibactam, imipenem/relebactam, and meropenem/vaborbactam were the least active options. Aztreonam/avibactam and aztreonam/nacubactam were active against the whole collection and yielded MIC50/MIC90 values of ≤0.25/0.5 mg/L and 1/2 mg/L, respectively. Cefepime/zidebactam (56/57 susceptible), meropenem/xeruborbactam (56/57 susceptible), cefepime/nacubactam (55/57 susceptible), and cefiderocol (53/57 susceptible) were also highly active, with MIC50/MIC90 values ranging from ≤0.25-2 mg/L to 2-4 mg/L, respectively. Meropenem/ANT3310 (MIC50/MIC90 = 0.5/≥64 mg/L; 47/57 susceptible) and cefepime/taniborbactam (MIC50/MIC90 = 0.5/16 mg/L; 44/57 susceptible) also retained high levels of activity, although they were affected by NDM-type enzymes in combination with porin deficiency. Our findings highlight that cefiderocol and combinations of β-lactams and the novel β-lactamase inhibitors avibactam, nacubactam, taniborbactam, zidebactam, xeruborbactam, and ANT3310 show promising activity against double-carbapenemase-producing Enterobacterales.

Assessment of Lefamulin 20 µg Disk versus Broth Microdilution When Tested against Common Respiratory Pathogens

ObjectiveTo evaluate the performance of the disk diffusion test with lefamulin 20 µg compared with the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution method. Methods572 clinical stains, including 240 Staphylococcus aureus, 211 Streptococcus pneumoniae and 121 Haemophilus influenzae, isolated from 71 medical centers from the China Antimicrobial Surveillance Network (CHINET) in 2020. Broth microdilution method and disk diffusion methods were performed according to CLSI. Categorical agreement (CA), major error (ME), and very major error (VME) were calculated. ResultsLefamulin showed potent activity against S. aureus, S. pneumoniae, and H. influenzae. Using the broth microdilution method, lefamulin inhibited 97.1% of S. aureus isolates at 0.25 mg/L; seven isolates were not susceptible. For S. pneumoniae and H. influenzae, the percentage of susceptibility to lefamulin was 100% and no non-susceptible strains were found in this study. Compared with the reference broth microdilution method, the CA of the lefamulin 20 µg disk testing was 99.8% (571/572), with 14.3% (1/7) VME and no ME. In our study, VME was determined in S. aureus. For S. pneumoniae and H. influenzae, the VME was not determined due to the lack of lefamulin non-susceptible strains. ConclusionThe lefamulin 20 µg disk diffusion testing showed excellent CA and ME with the reference broth microdilution method for S. aureus, S. pneumoniae, and H. influenzae. The VME exceeding CLSI recommendations may be a bias due to fewer lefamulin non-susceptible isolates. Our results suggest that lefamulin non-susceptible isolates detected by disk diffusion should be confirmed by the reference BMD.

Impact of transferable β-lactamases and intrinsic AmpC amino acid substitutions on the activity of cefiderocol against wild-type and iron uptake-deficient mutants of Pseudomonas aeruginosa.

We aimed to analyse the interplay between impaired iron uptake and β-lactamases on cefiderocol resistance in Pseudomonas aeruginosa. Thirty-one transferable β-lactamases and 16 intrinsic P. aeruginosa AmpC (PDC) variants were cloned and expressed in wild-type (PAO1) and iron uptake-deficient (PAO ΔpiuC) P. aeruginosa backgrounds. MICs of cefiderocol and antipseudomonal β-lactams were determined by reference broth microdilution. Relative to PAO1, deletion of piuC caused a specific 16-fold decrease in cefiderocol activity but negligible effects on the activity of other β-lactams. Among transferable β-lactamases, SHV-12, KPC Ω-loop mutants, NDMs and OXA-15 showed cefiderocol MIC values above the clinical breakpoint (2 mg/L) when expressed in PAO1. When expressed in PAO ΔpiuC, these and the transformants harbouring PER-1, VEB-1, KPC-2, KPC-3, VIM-1, CMY-2, OXA-2 and OXA-14 showed increased MIC values from 16 to >256 mg/L. The PDC variants carrying the Ω-loop changes ΔP215-G222 (PDC-577), E219K (PDC-221 and PDC-558) and the H10 helix change L293P (PDC-219) had the greatest impact on cefiderocol resistance, with MICs of 2-4 mg/L in PAO1 and of up to 32-64 mg/L in PAO ΔpiuC. Widespread enzymes such as GES, CTX-M-9, CTX-M-15, VIM-2-like enzymes, IMPs, DHA-1, FOX-4, OXA-10, OXA-48 and the other PDC variants tested had weaker effects on cefiderocol resistance. We add evidence about the effect of the interplay between iron uptake and β-lactamases on the acquisition of cefiderocol resistance in P. aeruginosa. These findings may help to anticipate the emergence of resistance and optimize the use of cefiderocol against P. aeruginosa infections.

Commercially available tests for determining cefiderocol susceptibility display variable performance in the Achromobacter genus

BackgroundCefiderocol is a siderophore-conjugated cephalosporin increasingly used in the management of Achromobacter infections. Testing for cefiderocol susceptibility is challenging with distinct recommendations depending on the pathogens.ObjectivesWe evaluated the performance of commercial tests for testing cefiderocol susceptibility in the Achromobacter genus and reviewed the literature.MethodsDiffusion (disks, MIC gradient test strips [MTS], Liofilchem) and broth microdilution (BMD) methods (ComASP™, Liofilchem; UMIC®, Bruker) were compared with the BMD reference method according to the EUCAST guidelines on 143 Achromobacter strains from 14 species with MIC50/90 of ≤ 0.015/0.5 mg/L. A literature search was conducted regardless of method or species.ResultsNone of the methods tested fulfilled an acceptable essential agreement (EA). MTS displayed the lowest EA (30.8%) after UMIC® (49%) and ComASP™ (76.9%). All methods achieved an acceptable bias, with MICs either underestimated using MTS (-1.3%) and ComASP™ (-14.2%) or overestimated with UMIC® (+ 9.1%). Inhibition zone diameters ranged from 6 to 38 mm (IZD50/90=33/30 mm). UMIC® and ComASP™ failed to categorize one or the two cefiderocol-resistant strains of this study as resistant unlike the diffusion-based methods. The literature review highlighted distinct performance of the available methods according to pathogens and testing conditions.ConclusionsThe use of MTS is discouraged for Achromobacter spp. Disk diffusion can be used to screen for susceptible strains by setting a threshold diameter of 30 mm. UMIC® and ComASP™ should not be used as the sole method but have to be systematically associated with disk diffusion to detect the yet rarely described cefiderocol-resistant Achromobacter sp. strains.

Erythromycin-metal complexes: One-step synthesis, molecular docking analysis and antibacterial proficiency against pathogenic strains

The study focused on the extraction of free erythromycin from commercially manufactured tablets and the use of metal salts to synthesize erythromycin-metal complexes, specifically involving silver (Ag), nickel (Ni), cobalt (Co), and copper (Cu). The synthesis was confirmed through various methods, including elemental analysis, thermogravimetric analysis, Fourier-transform infrared (FTIR), and UV–visible spectroscopy. The microbiological investigation involved Salmonella typhi, Escherichia coli, Staphylococcus aureus, Bacillus cereus, Candida albicans, and Microsporum canis as test organisms. The NCCLS broth microdilution reference method was used to determine the minimum fungicidal concentration and minimum inhibitory concentration of the complexes. The synthesized complexes were highly effective against a variety of fungi and bacteria, with compound Ery-Cu having MIC as low as 1.56 mg/mL, Ery-Cu and Ery-Ni with MBCs of 6.25 mg/mL and Ery-Cu having MFC of 6.25 mg/mL. Dose-dependent inhibitory effects were found upon examination of the antimicrobial susceptibility of specific complexes (Cu, Ni, Co and Ag) at varying concentrations of 100, 50, 25 and 12.5 mm/mL. Antibiotic susceptibility testing revealed efficacy against the tested pathogens. The study suggests that the synthesis of erythromycin-metal complexes, coupled with their antibacterial effectiveness against a diverse spectrum of bacteria and fungi, as they showed promising inhibitory properties when tested against a range of test species (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella typhi, Candida albicans, and Microsporum canis), could lead to the development of innovative antibacterial agents. Molecular docking simulations were used to examine the interactions between metal complexes with proteins filamentous temperature-sensitive protein Z and lanosterol 14α-demethylase. The study highlights the need for further exploration in pharmaceutical research.

Rapid detection of imipenem/relebactam susceptibility/resistance in Pseudomonas aeruginosa

ObjectivesImipenem-relebactam (IPR) has been reported to exhibit a good activity against non-metallo-ß-lactamase carbapenem-resistant Pseudomonas aeruginosa (CRPA), and the rapid detection of susceptibility/resistance to this new therapeutic alternative may be crucial. Therefore, the Rapid IPR Pseudomonas NP test was developed to quickly identify IPR susceptibility/resistance among multidrug-resistant P. aeruginosa. MethodsThe principle of the Rapid IPR Pseudomonas NP test is based on visually detecting glucose metabolization by observing (or not) a color change from yellow to red or orange of the red phenol pH indicator in the presence of imipenem at 2 mg/L and relebactam at 4 mg/L A total of 80 clinical Pseudomonas aeruginosa isolates were analyzed, among which 42 isolates were IPR resistant according to EUCAST guidelines (MICs, susceptible ≤2 mg/L, resistant >2 mg/L). Results obtained with the Rapid IPR Pseudomonas NP test were compared with the reference broth microdilution (BMD). ResultsThe sensitivity, specificity and accuracy of the test were found to be 100 %, 89.5 % and 95 %, respectively, using the BMD reference method as a comparator. Moreover, five out of the IPR-susceptible isolates (n = 38) exhibiting an MIC of IPR close to the breakpoint (MIC = 1 mg/L, n = 2; MIC = 2 mg/L, n = 3) yielded to a major error result, namely a positive result with the rapid IPR Pseudomonas NP test (resistance). By contrast, all IPR-resistant isolates (n = 42) were all correctly categorized. ConclusionsThe Rapid IPR Pseudomonas NP test is sensitive, specific, and easy to perform and interpret. Therefore, it is suitable for implementation in routine clinical laboratories.

Impact of chromosomally encoded resistance mechanisms and transferable β-lactamases on the activity of cefiderocol and innovative β-lactam/β-lactamase inhibitor combinations against Pseudomonas aeruginosa.

We aimed to compare the stability of the newly developed β-lactams (cefiderocol) and β-lactam/β-lactamase inhibitor combinations (ceftazidime/avibactam, ceftolozane/tazobactam, aztreonam/avibactam, cefepime/taniborbactam, cefepime/zidebactam, imipenem/relebactam, meropenem/vaborbactam, meropenem/nacubactam and meropenem/xeruborbactam) against the most clinically relevant mechanisms of mutational and transferable β-lactam resistance in Pseudomonas aeruginosa. We screened a collection of 61 P. aeruginosa PAO1 derivatives. Eighteen isolates displayed the most relevant mechanisms of mutational resistance to β-lactams. The other 43 constructs expressed transferable β-lactamases from genes cloned in pUCP-24. MICs were determined by reference broth microdilution. Cefiderocol and imipenem/relebactam exhibited excellent in vitro activity against all of the mutational resistance mechanisms studied. Aztreonam/avibactam, cefepime/taniborbactam, cefepime/zidebactam, meropenem/vaborbactam, meropenem/nacubactam and meropenem/xeruborbactam proved to be more vulnerable to mutational events, especially to overexpression of efflux operons. The agents exhibiting the widest spectrum of activity against transferable β-lactamases were aztreonam/avibactam and cefepime/zidebactam, followed by cefepime/taniborbactam, cefiderocol, meropenem/xeruborbactam and meropenem/nacubactam. However, some MBLs, particularly NDM enzymes, may affect their activity. Combined production of certain enzymes (e.g. NDM-1) with increased MexAB-OprM-mediated efflux and OprD deficiency results in resistance to almost all agents tested, including last options such as aztreonam/avibactam and cefiderocol. Cefiderocol and new β-lactam/β-lactamase inhibitor combinations show promising and complementary in vitro activity against mutational and transferable P. aeruginosa β-lactam resistance. However, the combined effects of efflux pumps, OprD deficiency and efficient β-lactamases could still result in the loss of all therapeutic options. Resistance surveillance, judicious use of new agents and continued drug development efforts are encouraged.

Antimicrobial susceptibility profile of ceftolozane/tazobactam, ceftazidime/avibactam and cefiderocol against carbapenem-resistant Pseudomonas aeruginosa clinical isolates from Türkiye.

Carbapenem resistant Pseudomonas aeruginosa (CR-PA) is escalating worldwide and leaves clinicians few therapeutic options in recent years, β-lactam/β-lactamase inhibitor combinations (ceftolozane-tazobactam, ceftazidime-avibactam) and a new siderophore cephalosporin (cefiderocol) have been approved for the treatment of P. aeruginosa infection and have shown potent activity against isolates defined as carbapenem resistant. The aim of this study was to determine the phenotypic profile of these agents against CR-PA in the emerging setting of carbapenemases. CR-PA clinical isolates were collected from three teaching hospitals in different geographical regions between January 2017-December 2021. All isolates were subjected to phenotypic carbapenemase testing using modified carbapenem inactivation method. MICs were determined by reference broth microdilution and evaluated according to EUCAST standards, while genotypic profiling was determined using PCR methods. 244 CR-PA sourced most frequently from the respiratory tract (32.2%), blood (20.4%) and urine (17.5%) were evaluated. Of all isolates, 32 (13.1%) were phenotypically and 38 (15.6%) were genotypically defined as carbapenemase-positive. The most common carbapenemase was GES (63.1%), followed by VIM (15.8%). The MIC50/90(S%) of ceftazidime/avibactam, ceftolozane/tazobactam and cefiderocol in all CR-PA isolates were 4 and 32 (80%), 1 and > 64 (69%) and 0.25 and 1mg/L (96%), respectively. Cefiderocol was also the most active agent in carbapenemase-positive isolates (90%). While ceftolozane/tazobactam and ceftazidime/avibactam remained highly active against CR-PA devoid of carbapenemases, cefiderocol provided potent in vitro activity irrespective of carbapenemase production. When considering the potential clinical utility of newer agents against CR-PA, regional variations in carbapenemase prevalence must be considered.

Cefiderocol susceptibility of Achromobacter spp.: study of an accurately identified collection of 230 strains

BackgroundAchromobacter spp. are opportunistic pathogens, mostly infecting immunocompromised patients and patients with cystic fibrosis (CF) and considered as difficult-to-treat pathogens due to both intrinsic resistance and the possibility of acquired antimicrobial resistance. Species identification remains challenging leading to imprecise descriptions of resistance in each taxon. Cefiderocol is a broad-spectrum siderophore cephalosporin increasingly used in the management of Achromobacter infections for which susceptibility data remain scarce. We aimed to describe the susceptibility to cefiderocol of a collection of Achromobacter strains encompassing different species and isolation sources from CF or non-CF (NCF) patients.MethodsWe studied 230 Achromobacter strains (67 from CF, 163 from NCF patients) identified by nrdA gene-based analysis, with available susceptibility data for piperacillin–tazobactam, meropenem and trimethoprim–sulfamethoxazole. Minimal inhibitory concentrations (MICs) of cefiderocol were determined using the broth microdilution reference method according to EUCAST guidelines.ResultsStrains belonged to 15 species. A. xylosoxidans represented the main species (71.3%). MICs ranged from ≤ 0.015 to 16 mg/L with MIC50/90 of ≤ 0.015/0.5 mg/L overall and 0.125/2 mg/L against 27 (11.7%) meropenem-non-susceptible strains. Cefiderocol MICs were not related to CF/NCF origin or species although A. xylosoxidans MICs were statistically lower than those of other species considered as a whole. Considering the EUCAST non-species related breakpoint (2 mg/L), 228 strains (99.1%) were susceptible to cefiderocol. The two cefiderocol-resistant strains (A. xylosoxidans from CF patients) represented 3.7% of meropenem-non-susceptible strains and 12.5% of MDR strains.ConclusionsCefiderocol exhibited excellent in vitro activity against a large collection of accurately identified Achromobacter strains, irrespective of species and origin.

Evaluation of commercial, customized microdilution plates for Ureaplasma parvum, Ureaplasma urealyticum, and Mycoplasma hominis antimicrobial susceptibility testing and determination of antimicrobial resistance prevalence in France.

Antimicrobial susceptibility testing of human urogenital mycoplasmas using the CLSI reference broth microdilution method is time-consuming and requires the laborious preparation of antimicrobial stock solutions. Here, we validated the use of reliable, convenient plates designed for antimicrobial susceptibility testing that allows the simultaneous determination of the MICs of eight antibiotics of interest. We then investigated the prevalence and mechanisms of resistance of each of these bacteria to tetracyclines, fluoroquinolones, and macrolides in France in 2020 and 2021. We showed that the prevalence of levofloxacin and moxifloxacin resistance has increased significantly in Ureaplasma spp. from 2010 to 2015 and requires ongoing monitoring.

Comparison of Colistin Susceptibility via Two Different Methods in Gram-Negative Extensive Drug-Resistance Isolates from ICU Patients.

To compare the susceptibility of colistin by two methods in extensive drug-resistant (XDR) Gram-negative isolates from ICU patients. Cross-sectional comparative analysis. Place and Duration of the Study: Department of Microbiology, Combined Military Hospital Karachi, Pakistan, from August 2022 to February2023. A total of 100 clinical specimens received from the intensive care unit yielded growth of extensively drug-resistant gram-negative bacteria, which were evaluated for polymyxin E susceptibility. The agar dilution method was compared with the reference broth microdilution (BMD) method. Minimum inhibitory concentration (MIC) was noted for both methods. Comparison of the MIC method by agar dilution showed a 90% correlation with the reference method of broth microdilution. With MICs within the acceptable range of the clinical and laboratory standards institute (CLSI) recommendations, 89 isolates were susceptible to colistin, whereas only 11 remained resistant. Polymyxin E's MIC 50 and MIC 90 were determined to be 1 and 2 µg/ml, respectively, with 97% susceptibility. Agar dilution susceptibility method can be used for screening purposes for the susceptibility testing of polymyxin E. This method is reliable and can easily identify the heteroresistance. Extensively drug-resistant, Broth microdilution, Multidrug-resistant, Agar dilution, Minimum inhibitory concentration, Colony forming unit.

Detection of KPC-216, a Novel KPC-3 Variant, in a Clinical Isolate of Klebsiella pneumoniae ST101 Co-Resistant to Ceftazidime-Avibactam and Cefiderocol.

Carbapenemase-producing Klebsiella pneumoniae (CP-KP) represents a global threat to public health, with limited antimicrobial therapeutic options. In this study, we analyzed a ceftazidime/avibactam (CAZ-AVI)-resistant K. pneumoniae isolate obtained from a patient previously exposed to CAZ-AVI expressing a novel K. pneumoniae carbapenemase (KPC)-3 variant. Antimicrobial susceptibility testing was performed using reference broth microdilution. Whole-genome sequencing (WGS) was performed using Illumina and Nanopore Technologies. Short- and long-reads were combined with Unicycler. Assemblies were investigated for multilocus sequence typing (MLST), antimicrobial resistance genes, porins, and plasmids. The K. pneumoniae isolate (KP_RM_1) was resistant to CAZ-AVI, expanded-spectrum cephalosporins, amikacin, ertapenem, and cefiderocol (FDC) but was susceptible to tigecycline, colistin, trimethoprim/sulfamethoxazole, meropenem-vaborbactam, and imipenem-relebactam. WGS revealed that the KP_RM_1 genome is composed of a single chromosome of 5 Mbp and five circular plasmids. Further analysis showed the presence of novel blaKPC-216 located on a 72 kb plasmid. KPC-216 differs from KPC-3 by a Lysin (K) insertion at position 168 (+K168). We report the identification of a new KPC-3 variant associated with CAZ-AVI resistance. The KPC variants associated with CAZ-AVI resistance should be determined to promptly inform clinicians and start the appropriate antimicrobial therapy.

Evaluation of a simple method for testing aztreonam and ceftazidime-avibactam synergy in New Delhi metallo-beta-lactamase producing Enterobacterales.

NDM-producing carbapenem-resistant bacterial infections became a challenge for clinicians. Combination therapy of aztreonam and ceftazidime-avibactam is a prudent choice for these infections. However, there is still no recommendation of a practically feasible method for testing aztreonam and ceftazidime-avibactam synergy. We proposed a simple method for testing aztreonam and ceftazidime-avibactam synergy and compared it with reference broth micro-dilution and other methods. Carbapenem-resistant Enterobacterales clinical isolates were screened for the presence of the NDM gene by the Carba R test. NDM harbouring isolates were tested for aztreonam and ceftazidime-avibactam synergy by broth microdilution (reference method), E strip-disc diffusion, double disc diffusion, and disc replacement methods. In the newly proposed method, the MHA medium was supplemented with ceftazidime-avibactam (corresponding to an aztreonam concentration of 4μg/ml). The MHA medium was then inoculated with the standard inoculum (0.5 McFarland) of the test organism. An AZT disc (30 μg) was placed on the supplemented MHA medium, and the medium was incubated overnight at 37°C. Aztreonam zone diameter on the supplemented MHA medium (in the presence of ceftazidime-avibactam) was compared with that from a standard disc diffusion plate (without ceftazidime-avibactam), performed in parallel. Interpretation of synergy was based on the restoration of aztreonam zone diameter (in the presence of ceftazidime-avibactam) crossing the CLSI susceptibility breakpoint, i.e., ≥ 21 mm. Of 37 carbapenem-resistant NDM-producing isolates, 35 (94.6%) were resistant to aztreonam and tested synergy positive by the proposed method. Its sensitivity and specificity were 97.14% and 100%, respectively. Cohen's kappa value showed substantial agreement of the reference method with the proposed method (κ = 0.78) but no other methods. The proposed method is simple, easily interpretable, and showed excellent sensitivity, specificity, and agreement with the reference method. Therefore, the new method is feasible and reliable for testing aztreonam synergy with avibactam in NDM-producing Enterobacterales.

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.

Performance of modified colistin broth disc elution vis-a-vis broth microdilution method for susceptibility testing of Enterobacterales

Objectives: Recently, Clinical and Laboratory Standards Institute (CLSI) has approved colistin broth disc elution (CBDE) to be a supplemental test. This requires multiple discs and tubes to get the desired concentrations of colistin -1, 2, and 4 µg/mL and 10 mL volume of cation-adjusted Mueller–Hinton broth for a single isolate. The present study was aimed to evaluate the performance of CBDE in a microtiter plate format modified (mCBDE) with the reference method broth microdilution (BMD) for detection of colistin resistance in carbapenem-resistant Enterobacterales (CRE) isolates. Materials and Methods: One hundred and sixty non-duplicate clinical CRE isolates (May 2021–April 2022) were simultaneously subjected for BMD and mCBDE. For mCBDE, colistin 10 µg discs and Mueller–Hinton broth no-2 control cations were procured from HiMedia, Mumbai, and drug concentrations were prepared following CLSI-M100Ed31. Results of mCBDE were compared with reference BMD (Minimum inhibitory concentration [MIC] ≤2 µg/mL – intermediate and ≥4 µg/mL – resistant). Statistical Analysis: The performance of mCBDE was compared with BMD and expressed in terms of Categorical, essential agreement (EA), very major error (VME), and major error (ME). The sensitivity and specificity were calculated using Fisher’s contingency Table. Results: Of the 160 CRE isolates, 152 had exactly the same minimal inhibitory concentration (MIC) in both the tests with four isolates having higher and four having lower colistin MIC by mCBDE, giving a major error of 2.1% and VME of 5.5%. Categorical and essential agreement of mCBDE were 97.5% and 98.7%, respectively. Conclusions: mCBDE is an easy, economical, and reliable alternative test for determining colistin susceptibility for CRE isolates. Further, large-scale study is needed to strengthen our observation.

Combining deep learning and droplet microfluidics for rapid and label-free antimicrobial susceptibility testing of colistin

Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains.

Activity of cefiderocol and innovative β-lactam/β-lactamase inhibitor combinations against isogenic strains of Escherichia coli expressing single and double β-lactamases under high and low permeability conditions

ObjectivesTo analyse the impact of the most clinically relevant β-lactamases and their interplay with low outer membrane permeability on the activity of cefiderocol, ceftazidime/avibactam, aztreonam/avibactam, cefepime/enmetazobactam, cefepime/taniborbactam, cefepime/zidebactam, imipenem/relebactam, meropenem/vaborbactam, meropenem/xeruborbactam and meropenem/nacubactam against recombinant Escherichia coli strains. MethodsWe constructed 82 E. coli laboratory transformants expressing the main β-lactamases circulating in Enterobacterales (70 expressing single β-lactamase and 12 producing double carbapenemase) under high (E. coli TG1) and low (E. coli HB4) permeability conditions. Antimicrobial susceptibility testing was determined by reference broth microdilution. ResultsAztreonam/avibactam, cefepime/zidebactam, cefiderocol, meropenem/xeruborbactam and meropenem/nacubactam were active against all E. coli TG1 transformants. Imipenem/relebactam, meropenem/vaborbactam, cefepime/taniborbactam and cefepime/enmetazobactam were also highly active, but unstable against most of MBL-producing transformants. Combination of β-lactamases with porin deficiency (E. coli HB4) did not significantly affect the activity of aztreonam/avibactam, cefepime/zidebactam, cefiderocol or meropenem/nacubactam, but limited the effectiveness of the rest of carbapenem- and cefepime-based combinations. Double-carbapenemase production resulted in the loss of activity of most of the compounds tested, an effect particularly evident for those E. coli HB4 transformants in which MBLs were present. ConclusionsOur findings highlight the promising activity that cefiderocol and new β-lactam/β-lactamase inhibitors have against recombinant E. coli strains expressing widespread β-lactamases, including when these are combined with low permeability or other enzymes. Aztreonam/avibactam, cefiderocol, cefepime/zidebactam and meropenem/nacubactam will help to mitigate to some extent the urgency of new compounds able to resist MBL action, although NDM enzymes represent a growing challenge against which drug development efforts are still needed.

Resistance Profile, Terbinafine Resistance Screening and MALDI-TOF MS Identification of the Emerging Pathogen Trichophyton indotineae

The emerging pathogen Trichophyton indotineae, often resistant to terbinafine (TRB), is known to cause severe dermatophytoses such as tinea corporis and tinea cruris. In order to achieve successful treatment for these infections, insight in the resistance profile of T. indotineae strains and rapid, reliable identification is necessary. In this research, a screening medium was tested on T. indotineae strains (n = 20) as an indication tool of TRB resistance. The obtained results were confirmed by antifungal susceptibility testing (AST) for TRB following the in vitro broth microdilution reference method. Additionally, AST was performed for eight other antifungal drugs: fluconazole, itraconazole, voriconazole, ketoconazole, griseofulvin, ciclopirox olamine, naftifine and amorolfine. Forty-five percent of the strains were confirmed to be resistant to terbinafine. The TRB resistant strains showed elevated minimal inhibitory concentration values for naftifine and amorolfine as well. DNA sequencing of the squalene epoxidase-encoding gene showed that TRB resistance was a consequence of missense point mutations in this gene, which led to amino acid substitutions F397L or L393F. MALDI-TOF MS was used as a quick, accurate identification tool for T. indotineae, as it can be challenging to distinguish it from closely related species such as Trichophyton mentagrophytes or Trichophyton interdigitale using morphological characteristics. While MALDI-TOF MS could reliably identify ≥ 95% of the T. indotineae strains (depending on the spectral library), it could not be used to successfully distinguish TRB susceptible from TRB resistant strains.

Rates of resistance and heteroresistance to newer β-lactam/β-lactamase inhibitors for carbapenem-resistant Enterobacterales.

Heteroresistance (HR), the presence of antibiotic-resistant subpopulations within a primary isogenic population, may be a potentially overlooked contributor to newer β-lactam/β-lactamase inhibitor (BL/BLI) treatment failure in carbapenem-resistant Enterobacterales (CRE) infections. To determine rates of susceptibility and HR to BL/BLIs ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam in clinical CRE isolates. The first CRE isolate per patient per year from two >500 bed academic hospitals from 1 January 2016 to 31 December 2021, were included. Reference broth microdilution (BMD) was used to determine antibiotic susceptibility, and population analysis profiling (PAP) to determine HR. Carbapenemase production (CP) was determined using the Carba NP assay. Among 327 CRE isolates, 46% were Enterobacter cloacae, 38% Klebsiella pneumoniae and 16% Escherichia coli. By BMD, 87% to 98% of CRE were susceptible to the three antibiotics tested. From 2016 to 2021, there were incremental decreases in the rates of susceptibility to each of the three BL/BLIs. HR was detected in each species-antibiotic combination, with the highest rates of HR (26%) found in K. pneumoniae isolates with imipenem/relebactam. HR or resistance to at least one BL/BLI by PAP was found in 24% of CRE isolates and 65% of these had detectable CP. Twenty-four percent of CRE isolates tested were either resistant or heteroresistant (HR) to newer BL/BLIs, with an overall decrease of ∼10% susceptibility over 6 years. While newer BL/BLIs remain active against most CRE, these findings support the need for ongoing antibiotic stewardship and a better understanding of the clinical implications of HR in CRE.