Minimum Inhibitory Concentration Distributions Research Articles

662. In vitro antibacterial activity of contezolid compared to linezolid and vancomycin in clinical isolates from China

Abstract Background Linezolid and vancomycin are insufficient in the treatment of drug-resistant Gram-positive bacteria due to adverse drug reactions and drug resistance, and new antibiotics are needed, especially for patients with myelosuppression and vancomycin-resistant Enteroccocus (VRE) infection. Contezolid (CZD) is a novel oral oxazolidinone with potent activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and VRE. We evaluate the in vitro antibacterial activity of contezolid compared to linezolid and vancomycin in Gram-positive clinical isolates. Methods 176 clinical isolates were obtained from 176 sterile body fluids from patients at 3 tertiary care hospitals in China, and the minimal inhibitory concentrations (MICs) of contezolid, linezolid and vancomycin determined by broth microdilution assay. Results Contezolid exhibits excellent antibacterial activity against prevalent Gram-positive bacteria, Staphylococcus spp. and Enterococcus spp., including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant and coagulase-negative Staphylococcus (MRCNS), and vancomycin-resistant Enterococcus (VRE). MIC90 values of contezolid were 2 mg/L for all MRSA and VRE isolates and 1 mg/L for MRCNS isolates. The antibacterial activity of contezolid against Staphylococcus spp., including S. aureus, was essentially similar to linezolid, and slightly higher than or similar to vancomycin. Antibacterial activity of contezolid against Enterococcus, including VRE, was similar to linezolid, while being slightly lower than for vancomycin in vancomycin-susceptible isolates. Consistent with the shared antibacterial mode of action, bacterial strains resistant to linezolid were cross-resistant to contezolid. Table 1.Antimicrobial susceptibility testing results of clinical bacterial strains (MICs, mg/L) Conclusion Contezolid, a novel oxazolidinone drug, exhibits potent in vitro antibacterial activity against clinical isolates of common Gram-positive pathogens, including multidrug-resistant (MDR) Gram-positive bacteria, MRSA, MRCNS, and VRE. Contezolid has antibacterial activity similar to standard-of-care antibiotics linezolid and vancomycin. Results suggest that contezolid can play an important role in treatment of MDR Gram-positive bacterial infections. Figure 1.Distribution of MICs of CZD or LZD against 48 strains of MRSAFigure 2.Distribution of MICs of CZD or LZD against 29 strains of MRCNSFigure 3.Distribution of MICs of CZD or LZD against 17 strains of VRE Disclosures All Authors: No reported disclosures.

The Inappropriateness of Using Rifampicin E-Test to Predict Rifabutin Resistance in Helicobacter pylori

The aim of this study was to evaluate the rifamycin cross-resistance in Helicobacter pylori, and whether the use of rifampicin E-test strips to screen H. pylori rifabutin resistance is appropriate. A total of 89 H. pylori isolates were included. Rifampicin minimum inhibitory concentrations (MICs) were obtained by E-test, while the MICs for rifapentine, rifaximin, and rifabutin were determined by agar dilution method. The rifamycin resistance rates based on different breakpoints were compared. Isolates with high-level rifampicin resistance were subjected to whole-genome sequencing. A wide distribution of MICs (mostly in the range 0.125-8 mg/L) was observed for rifampicin, rifapentine, and rifaximin. Using MIC >1, ≥ 4, and > 4 mg/L as the breakpoints, resistance rates to rifampicin/rifapentine/rifaximin were 60.4%/48.3%/38.2%, 28.1%/25.8%/23.6%, and 15.7%/16.9%/7.9%, respectively. However, the rifabutin MICs of all the tested H. pylori isolates were extremely low (≤0.016 mg/L). Applying MIC ≥ 0.125 mg/L as the breakpoint, rifabutin resistance was nil. No mutation was found in the rpoB gene sequences of the 2 isolates with high-level rifampicin resistance. There is a lack of cross-resistance between rifabutin and other rifamycins in H. pylori. The use of rifampicin E-test to predict H. pylori rifabutin resistance is inappropriate.

Recent Trend of Antimicrobial Susceptibility among Mycoplasma pneumoniae Isolated from Japanese Children.

Macrolide-resistant Mycoplasma pneumoniae (MRMP) infections have become increasingly prevalent, especially in East Asia. Whereas MRMP strains have point mutations that are implicated in conferring resistance, monitoring the antibiotic susceptibility of M. pneumoniae and identifying mutations in the resistant strains is crucial for effective disease management. Therefore, we investigated antimicrobial susceptibilities among M. pneumoniae isolates obtained from Japanese children since 2011. To establish the current susceptibility trend, we analyzed the minimum inhibitory concentrations (MICs) of M. pneumoniae in recent years (2017-2020) in comparison with past data. Our observation of 122 M. pneumoniae strains suggested that 76 were macrolide-susceptible M. pneumoniae (MSMP) and 46 were macrolide-resistant. The MIC ranges (µg/mL) of clarithromycin (CAM), azithromycin (AZM), tosufloxacin (TFLX), and minocycline (MINO) to all M. pneumoniae isolates were 0.001->128, 0.00012->128, 0.25-0.5, and 0.125-4 µg/mL, respectively. None of the strains was resistant to TFLX or MINO. The MIC distributions of CAM and AZM to MSMP and MINO to all M. pneumoniae isolates were significantly lower, but that of TFLX was significantly higher than that reported in all previous data concordant with the amount of recent antimicrobial use. Therefore, continuation of appropriate antimicrobial use for M. pneumoniae infection is important.

A multicategory logit model detecting temporal changes in antimicrobial resistance.

Monitoring and investigating temporal trends in antimicrobial data is a high priority for human and animal health authorities. Timely detection of temporal changes in antimicrobial resistance (AMR) can rely not only on monitoring and analyzing the proportion of resistant isolates based on the use of a clinical or epidemiological cut-off value, but also on more subtle changes and trends in the full distribution of minimum inhibitory concentration (MIC) values. The nature of the MIC distribution is categorical and ordinal (discrete). In this contribution, we developed a particular family of multicategory logit models for estimating and modelling MIC distributions over time. It allows the detection of a multitude of temporal trends in the full discrete distribution, without any assumption on the underlying continuous distribution for the MIC values. The experimental ranges of the serial dilution experiments may vary across laboratories and over time. The proposed categorical model allows to estimate the MIC distribution over the maximal range of the observed experiments, and allows the observed ranges to vary across labs and over time. The use and performance of the model is illustrated with two datasets on AMR in Salmonella.

Effect of povidone-iodine and propanol-based mecetronium ethyl sulphate on antimicrobial resistance and virulence in Staphylococcus aureus

BackgroundReports are available on cross-resistance between antibiotics and biocides. We evaluated the effect of povidone-iodine (PVP-I) and propanol-based mecetronium ethyl sulphate (PBM) on resistance development, antibiotics cross-resistance, and virulence in Staphylococcus aureus.MethodsThe minimum inhibitory concentration (MIC) of PVP-I and PBM were determined against S. aureus ATCC 25923 using the agar-dilution method. Staphylococcus aureus ATCC 25923 was subjected to subinhibitory concentrations of the tested biocides in ten consecutive passages followed by five passages in a biocide-free medium; MIC was determined after each passage and after the fifth passage in the biocide-free medium. The developed resistant mutant was tested for cross-resistance to different antibiotics using Kirby-Bauer disk diffusion method. Antibiotic susceptibility profiles as well as biocides’ MIC were determined for 97 clinical S. aureus isolates. Isolates were categorized into susceptible and resistant to the tested biocides based on MIC distribution pattern. The virulence of the biocide-resistant mutant and the effect of subinhibitory concentrations of biocides on virulence (biofilm formation, hemolysin activity, and expression of virulence-related genes) were tested.ResultsPVP-I and PBM MIC were 5000 μg/mL and 664 μg/mL. No resistance developed to PVP-I but a 128-fold increase in PBM MIC was recorded, by repeated exposure. The developed PBM-resistant mutant acquired resistance to penicillin, cefoxitin, and ciprofloxacin. No clinical isolates were PVP-I-resistant while 48.5% were PBM-resistant. PBM-resistant isolates were more significantly detected among multidrug-resistant isolates. PVP-I subinhibitory concentrations (¼ and ½ of MIC) completely inhibited biofilm formation and significantly reduced hemolysin activity (7% and 0.28%, respectively). However, subinhibitory concentrations of PBM caused moderate reduction in biofilm activity and non-significant reduction in hemolysin activity. The ½ MIC of PVP-I significantly reduced the expression of hla, ebps, eno, fib, icaA, and icaD genes. The virulence of the biocide-resistant mutant was similar to that of parent strain.ConclusionPVP-I is a highly recommended antiseptic for use in healthcare settings to control the evolution of high-risk clones. Exposure to PVP-I causes no resistance-development risk in S. aureus, with virulence inhibition by subinhibitory concentrations. Also, special protocols need to be followed during PBM use in hospitals to avoid the selection of resistant strains.

Determination of critical concentration for drug susceptibility testing of Mycobacterium tuberculosis against para-aminosalicylic acid with clinical isolates with thyA, folC and dfrA mutations

Background & ObjectivesAccurate determination of antimicrobial resistance profiles is of great importance to formulate optimal regimens against multidrug-resistant tuberculosis (MDR-TB). Although para-aminosalicylic acid (PAS) has been widely used clinically, the reliable testing methods for PAS susceptibility were not established. Herein, we aimed to establish critical test concentration for PAS on the Mycobacterial Growth Indicator Tube (MGIT) 960 in our laboratory settings.MethodsA total of 102 clinical isolates were included in this study, including 82 wild-type and 20 resistotype isolates. Minimum inhibitory concentration (MIC) was determined by MGIT 960. Whole-genome sequencing was used to identify the mutation patterns potentially conferring PAS resistance. Sequence alignment and structure modelling were carried out to analyze potential drug-resistant mechanism of folC mutant.ResultsOverall, the Minimum inhibitory concentration (MIC) distribution demonstrated excellent separation between wild-type and resistotype isolates. The wild-type population were all at least 1 dilution below 4 μg/ml, and the resistotype population were no lower than 4 μg/ml, indicating that 4 μg/ml was appropriate critical concentration to separate these two populations. Of 20 mutant isolates, 12 (60.0%) harbored thyA mutations, 2 (10%) had a mutation on upstream of dfrA, and the remaining isolates had folC mutations. Overall, thyA and folC mutations were scattered throughout the whole gene without any one mutation predominating. All mutations within thyA resulted in high-level resistance to PAS (MIC > 32 μg/ml); whereas the MICs of isolates with folC mutations exhibited great diversity, ranged from 4 to > 32 μg/ml, and sequence and structure analysis partially provided the possible reasons for this diversity.ConclusionsWe propose 4 μg/ml as tentative critical concentration for MGIT 960. The major mechanism of PAS resistance is mutations within thyA and folC in MTB isolations. The whole-gene deletion of thyA locus confers high-level resistance to PAS. The diversity of many distinct mutations scattered throughout the full-length folC gene challenges the PCR-based mutation analysis for PAS susceptibility.

Analysis of In Vitro Activity of Cefiderocol Against Carbapenem-Resistant Gram-Negative Bacilli by Broth Microdilution and Disk Diffusion Method: A Single-Center Study in Odisha, India

IntroductionCefiderocol (CFDC), a novel semi-synthetic siderophore cephalosporin has been developed to combat the menace of infections caused by carbapenem-resistant Gram-negative bacilli (CR-GNB) including Carbapenem-resistant Enterobacterales (CRE) and Carbapenem-resistant Nonfermenting Gram-negative bacilli (CR-NFGNB).MethodsWe determined the in vitro activity of CFDC against a contemporary collection of 503 CR-GNB isolates by the reference broth microdilution method (BMD) using Iron depleted cation adjusted Mueller-Hinton broth (ID-CAMHB). Performance of CFDC disk diffusion (DD) was evaluated against the reference BMD, as an alternative convenient testing method. Molecular characterization of carbapenemase in CR-GNB was performed by PCR targeting blaNDM-1, blaOXA-48like alleles, blaKPC, blaIMP, and, blaVIM. Minimum inhibitory concentration (MIC) distribution of CFDC in CR-GNB harbouring different carbapenemase enzymes was also analyzed.ResultsIn our study, 81.7% (411/503) of CR-GNB isolates [81.3%, (278/342) CRE and 82.6% (133/161) CR-NFGNB] were susceptible to CFDC (p>0.05). Categorical agreement (CA) of DD ranged from 79.8% to 87.5%, Minor error (mE) ranged from 0 to 14%, Major error (ME) ranged from 0 to 3.5%, and Very Major error (VME) ranged from 0 to 12.5% with variations by species tested. Overall CFDC MIC50 and MIC90 values of CR-GNB isolates without any carbapenemase genes were higher as compared to those with the presence of carbapenemase genes (4 µg/mL and 128 µg/mL versus 2 µg/mL and 16 µg/mL respectively).DiscussionCFDC is not yet available for clinical use in India. Hence, multicentric studies are the need of the hour in India for standardization of CFDC susceptibility using disks and CAMHB from different manufacturers as well as understanding mechanisms of high MIC values.

Phenotypic and genotypic antibiotic resistance patterns in Salmonella Typhimurium and its monophasic variant from pigs in southern Spain

The high incidence of human salmonellosis and multi-drug resistant (MDR) strains of Salmonella Typhimurium (ST) is of concern to global public and animal health. Our research, by means of the broth microdilution method, evaluated the Minimum Inhibitory Concentration (MIC) distribution of 12 antimicrobials against a collection of 73 ST and mST and S. typhimurium monophasic variant 4,[5],12:i:- (mST) isolates from slaughtered pigs reared in extensive systems in southern Spain, and also 12 resistance-associated genes or antimicrobial resistance (AMR) determinants using qPCR.Our data revealed that 98.6% of strains were MDR, with resistance to cephalothin/tetracycline/sulfamethoxazole-trimethoprim/ampicillin/chloramphenicol being the most common pattern (55.6%). Regarding AMR determinants, the most significantly (p < 0.05) genes detected by qPCR were sul1 and aadA2 (89% of strains positive), aadA1 and dfrA12 (87.7%), and blaTEM and tet(B) (86.3% and 84.9%, respectively).Up to date information on ST antimicrobial resistance patterns is essential for epidemiological surveillance programs to support animal and public health. The high number of MDR isolates and variability regarding resistance determinants revealed in this study highlights the role of animals reared in extensive systems as a source of resistant Salmonella strains.

S4.5c Using serum beta-glucan measurements and sequencing of the Madurella mycetomatis azole target gene to predict therapeutic outcome during azole treatment in human mycetoma

S4.5 Mycetoma Clinical Trial on fosravuconazole treatment in eumycetoma– Top Line Results, September 22, 2022, 10:30 AM - 12:00 PMObjectivesEumycetoma is a neglected tropical disease characterized by large subcutaneous swellings and the formation of grains and most commonly caused by Madurella mycetomatis. The currently recommended therapy is a combination of antifungal therapy with an azole and surgery. Itraconazole is the current recommended drug and fosravuconazole, the pro-drug of ravuconzole, is currently clinically investigated. At the moment, there are no epidemiological cut-off values (ECV) for M. mycetomatis for either of these drugs or rapid diagnostic tests which can predict the therapeutic outcome of these treatments. Therefore, in this study, we determined the ECV for these drugs and determined whether there was a correlation between minimal inhibitory concentration (MIC) and the DNA sequence of the azole target gene CYP51A. We also assessed beta-glucan concentrations in the serum of mycetoma patients during treatment to establish whether any of these values were predictive for therapeutic outcomes.MethodsIn order to determine the ECV for M. mycetomatis, MIC distributions for itraconazole and ravuconazole were determined in genetically diverse clinical M. mycetomatis isolates using the ECOFFinder software. CYP51A sequences were sequenced and comparisons were made between the different CYP51A variants and the MIC distributions. Beta-glucan concentrations were measured in serum with the WAKO beta-glucan assay. Time points analyzed were 0, 22, 85, 176, 267, 358, and 455 days after the start of treatment.ResultsFor M. mycetomatis the MICs ranged from 0.008 to 1 mg/l for itraconazole and from 0.002 to 0.125 mg/l for ravuconazole. The M. mycetomatis ECV for itraconazole was 1 mg/l and for ravuconazole 0.064 mg/l. In the wild-type population, two CYP51A variants were found for M. mycetomatis, which differed in one amino acid at position 499. The MIC distributions for itraconazole and ravuconazole were similar between the two variants. No mutations linked to decreased susceptibility were found. Before the start of treatment, beta-glucan concentrations ranged from below the detection limit to 217.9 pg/ml. Of these patients, 61.2% had a beta-glucan concentration above 7 pg/ml, the recommended cut-off value for positivity by the manufacturer, 72.8% had a beta-glucan concentration above 5.5 pg/ml, the recommended cut-off value for M. mycetomatis. During the first months of azole treatment, the beta-glucan concentrations remained relatively stable. After surgery, a sharp decrease in beta-glucan concentration in serum was noted. At the end of the observation period, only 13 patients had a beta-glucan concentration above 7 pg/ml and 14 above 5.5 pg/ml. Of these patients, for only 3, there was clinical evidence of a recurrence. For the remaining 4 patients with clinical evidence of a recurrence, the beta-glucan concentration was below the cut-off value for positivity.ConclusionIn conclusion, so far there was no link established with the initial in vitro susceptibility and failure or success of the treatment therapy. Beta-glucan levels, in general, remained high during azole treatment, and a sharp drop in beta-glucan concentration in serum was only noted after surgery. A positive beta-glucan concentration at the end of the treatment was not indicative of a recurrence.

S4.1d In vitro activity of eight antifungals against 3343 filamentous pathogenic fungi collected during 18 years at the French National Reference Center for invasive mycosis and antifungals

S4.1 Treatment of rare mold infections in 2021: the role of new and old antifungals, September 22, 2022, 10:30 AM - 12:00 PM Background & ObjectivesThe NRCMA oversees the surveillance of invasive fungal diseases in France. As part of our expertise, we perform antifungal susceptibility testing based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology on all isolates. The antifungal profiles help us monitor the emergence of resistant isolates, determine the susceptibility pattern of wild-type strains to new antifungals, and in specific cases, determine the relationship between clinical failure and selection/emergence of a less susceptible isolate.We review the Minimum inhibitory concentrations (MIC) distribution of eight antifungals on clinical isolates of filamentous fungi identified at the NRCMA from 2003 to 2021.MethodsSpecies identification was performed by a combination of morphological features and multilocus sequencing. Only strains that produced enough conidia or spores were tested. In vitro susceptibility testing was performed according to the EUCAST procedure. Eight antifungal agents were used: triazoles [itraconazole, voriconazole, posaconazole, isavuconazole (since 2015)], echinocandins (caspofungin, micafungin), amphothericin B, and terbinafine. The concentrations inhibiting 50% (MIC50) and 90% (MIC90) of the isolates were determined for species with at least 5 and 10 isolates, respectively. For Aspergillus fumigatus isolates exhibiting high azole MICs, we sequenced the cyp51A gene for mutation screening.ResultsMICs were obtained for 3343 pathogenic strains. We identified Aspergillus spp. including cryptic species (32%), Fusarium spp. (21%), Mucorales (18%), phaeohyphomycetes (10%), i.e., Alternaria spp., Curvularia spp., Fonsecaea, Exophiala, Phaeoacremonium, rare hyphomycetes (9%), i.e., Rasamsonia, Paecilomyces, Trichoderma, Scopulariopsis. Scedosporium spp. (7%) and emergent pathogens such as Nannizziopsis obscura (1%), dimorphic fungi (1%), or to a less extent basidiomycetous molds. Examples of MIC results are presented in Table 1.For the nine genera of the order Mucorales, amphotericin B (AmB) exhibited low MICs except for Cunninghamella species and Saksenaea vasiformis. Posaconazole had variable activity depending on the species (high MIC values were observed for Mucor species) while voriconazole and echinocandins had none.In section Fumigati, amphotericin B MIC values were high due to the intrinsic resistance of cryptic species Aspergillus lentulus and A. udagawae. Other cryptic species were identified such as A. hiratsukae isolates which exhibited low MICs to all antifungals.Members of Aspergillus section Nidulantes such as A. nidulans and A. quadrilineatus showed variable MICs to caspofungin and amphotericin B.Among melanized fungi, Alternaria infectoria and A. alternata species group displayed low MICs of all azoles with the exception of voriconazole.Four Exophiala species were analyzed and had similar azole MICs, E. spinifera being the species with the lowest values.Multi-drug resistant profiles were observed especially in species belonging to Microascales such as Lomentospora prolificans, Microascus cirrosus, and Scopulariopsis brevicaulis. Members of Fusarium species complexes had predictably high MIC values to all antifungals including isavuconazole. Only Fusarium dimerum species complex had low MICs to amphotericin B.Further distribution analysis is ongoing. These large datasets provide a baseline for monitoring the emergence of antifungal resistance in our country and supports the fact that MIC determination should be performed for rare/emergent species and also in case of infections due to cryptic species, which would benefit patient management.

Antimicrobial susceptibility and minimum inhibitory concentration distribution of common clinically relevant non-tuberculous mycobacterial isolates from the respiratory tract

Objective: To determine the minimum inhibitory concentration (MIC) distribution of antibacterial drugs and the susceptibility of non-tuberculous mycobacterial (NTM) isolates to provide a reference basis for the clinical selection of an effective starting regimen. Methods: The common clinical isolates of NTM in the respiratory tract, which met the standards of the American Thoracic Society for NTM lung disease, were collected. The MICs of 81 isolates were determined using the microbroth dilution method (Thermo Fisher Scientific, USA), as recommended by the Clinical and Laboratory Standards Institute, USA. Results: Included were 43 Mycobacterium avium complex (MAC) strains, 24 M. abscessus complex (MAB) strains, and 14 M. kansasii strains. The sensitivity rates of MAC to clarithromycin and amikacin were 81.4% and 79.1%, respectively, while the sensitivity rates to linezolid and moxifloxacin were only 20.9% and 9.3%; the MIC of rifabutin was the lowest (MIC50% was just 2 μg/mL). After incubation for 3–5 days, the sensitivity rate of MAB to clarithromycin was 87.5%; this decreased to 50% after 14 days’ incubation. Most of them were susceptible to amikacin (91.6%), and most were resistant to moxifloxacin (95.8%), ciprofloxacin (95.8%), imipenem (95.8%), amoxicillin/clavulanate (95.8%), tobramycin (79.1%), doxycycline (95.8%) and trimethoprim/sulfamethoxazole (95.8%). intermediate (83.3%) and resistant (16.7%) to cefoxitin. The susceptibility to linezolid was only 33.3%. The sensitivity and resistance breakpoints of tigecycline were set to ≤0.5 and ≥8 μg/mL, respectively, and the sensitivity and resistance rates were 50% and 0%, respectively. M. kansasii was susceptible to clarithromycin, amikacin, linezolid, moxifloxacin, rifampicin and rifabutin (100%). Discussion: In Wenzhou, clarithromycin, amikacin and rifabutin have good antibacterial activity against MAC, while linezolid and moxifloxacin have high resistance. Amikacin and tigecycline have strong antibacterial activity against MAB, while most other antibacterial drugs are resistant to varying degrees. Most antibacterial drugs are susceptible to M. kansasii and have good antibacterial activity. Conclusion: The identification of NTM species and the detection of their MICs have certain guiding values for the treatment of NTM lung disease. Key Message The three most common respiratory non-tuberculous mycobacterial (NTM) isolates with clinical significance in the Wenzhou area were tested for drug susceptibility. The broth microdilution method was used to determine the minimum inhibitory concentration distribution of antibacterial drugs and the susceptibility of NTM isolates to provide a reference basis for the clinical selection of an effective starting regimen.

Epidemiological cut-off values for itraconazole and ravuconazole for Madurella mycetomatis, the most common causative agent of mycetoma.

Eumycetoma is a neglected tropical disease. It is a chronic inflammatory subcutaneous infection characterised by painless swellings which produce grains. It is currently treated with a combination of itraconazole and surgery. In an ongoing clinical study, the efficacy of fosravuconazole, the prodrug of ravuconazole, is being investigated. For both itraconazole and ravuconazole, no clinical breakpoints or epidemiological cut-off values (ECV) to guide treatment are currently available. To determine tentative ECVs for itraconazole and ravuconazole in Madurella mycetomatis, the main causative agent of eumycetoma. Minimal inhibitory concentrations (MICs) for itraconazole and ravuconazole were determined in 131 genetically diverse clinical M. mycetomatis isolates with the modified CLSI M38 broth microdilution method. The MIC distributions were established and used to determine ECVs with the ECOFFinder software. CYP51A sequences were sequenced to determine whether mutations occurred in this azole target gene, and comparisons were made between the different CYP51A variants and the MIC distributions. The MICs ranged from 0.008 to 1mg/L for itraconazole and from 0.002 to 0.125 mg/L for ravuconazole. The M. mycetomatis ECV for itraconazole was 1mg/L and for ravuconazole 0.064 mg/L. In the wild-type population, two CYP51A variants were found for M. mycetomatis, which differed in one amino acid at position 499 (S499G). The MIC distributions for itraconazole and ravuconazole were similar between the two variants. No mutations linked to decreased susceptibility were found. The proposed M. mycetomatis ECV for itraconazole is 1mg/L and for ravuconazole 0.064 mg/L.

Antimicrobial susceptibility profiles and tentative epidemiological cutoff values of Legionella pneumophila from environmental water and soil sources in China.

Legionnaires’ disease (LD), caused by Legionella, including the most prevalent Legionella pneumophila, has been treated primarily with antibiotics. Environmental water and soil are the reservoirs for L. pneumophila. Studying antimicrobial susceptibility using a large number of isolates from various environmental sources and regions could provide an unbiased result. In the present study, antimicrobial susceptibility of 1464 environmental L. pneumophila isolates that were derived from various environmental water and soil sources of 12 cities in China to rifampin (RIF), erythromycin (ERY), clarithromycin (CLA), azithromycin (AZI), ciprofloxacin (CIP), moxifloxacin (MOX), levofloxacin (LEV), and doxycycline (DOX) was investigated, and minimum inhibitory concentration (MIC) data were obtained. We show that regarding macrolides, ERY was least active (MIC90 = 0.5 mg/L), while CLA was most active (MIC90 = 0.063 mg/L). A total of three fluoroquinolones have similar MICs on L. pneumophila. Among these antimicrobials, RIF was the most active agent, while DOX was the most inactive one. We observed different susceptibility profiles between serogroup 1 (sg1) and sg2-15 or between water and soil isolates from different regions. The ECOFFs were ERY and AZI (0.5 mg/L), RIF (0.002 mg/L), CIP, CLA and MOX (0.125 mg/L), LEV (0.063 mg/), and DOX (32 mg/L). Overall, two fluoroquinolone-resistant environmental isolates (0.14%) were first documented based on the wild-type MIC distribution. Not all azithromycin-resistant isolates (44/46, 95.65%) harbored the lpeAB efflux pump. The MICs of the ERY and CLA on the lpeAB + isolates were not elevated. These results suggested that the lpeAB efflux pump might be only responsible for AZI resistance, and undiscovered AZI-specific resistant mechanisms exist in L. pneumophila. Based on the big MIC data obtained in the present study, the same defense strategies, particularly against both CLA and RIF, may exist in L. pneumophila. The results determined in our study will guide further research on antimicrobial resistance mechanisms of L. pneumophila and could be used as a reference for setting clinical breakpoints and discovering antimicrobial-resistant isolates in the clinic, contributing to the antibiotic choice in the treatment of LD.

Pharmacodynamic Target Assessment and PK/PD Cutoff Determination for Gamithromycin Against Streptococcus suis in Piglets.

Gamithromycin is a long-acting azalide antibiotic that has been developed recently for the treatment of swine respiratory diseases. In this study, the pharmacokinetic/pharmacodynamic (PK/PD) targets, PK/PD cutoff, and optimum dosing regimen of gamithromycin were evaluated in piglets against Streptococcus suis in China, including a subset with capsular serotype 2. Short post-antibiotic effects (PAEs) (0.5–2.6 h) and PA-SMEs (2.4–7.7 h) were observed for gamithromycin against S. suis. The serum matrix dramatically facilitated the intracellular uptake of gamithromycin by S. suis strains, thus contributing to the potentiation effect of serum on their susceptibilities, with a Mueller-Hinton broth (MHB)/serum minimum inhibitory concentration (MIC) ratio of 28.86 for S. suis. Dose-response relationship demonstrated the area under the concentration (AUC)/MIC ratio to be the predictive PK/PD index closely linked to activity (R2 > 0.93). For S. suis infections, the net stasis, 1–log10, and 2–log10 kill effects were achieved at serum AUC24h/MIC targets of 17.9, 49.1, and 166 h, respectively. At the current clinical dose of 6.0 mg/kg, gamithromycin PK/PD cutoff value was determined to be 8 mg/L. A PK/PD-based dose assessment demonstrated that the optimum dose regimen of gamithromycin to achieve effective treatments for the observed wild-type MIC distribution of S. suis in China with a probability of target attainment (PTA) ≥ 90% was 2.53 mg/kg in this study. These results will aid in the development of clinical dose-optimization studies and the establishment of clinical breakpoints for gamithromycin in the treatment of swine respiratory infections due to S. suis.

Pseudomonas aeruginosa susceptibility, antibiogram and clinical interpretation, and antimicrobial prescribing behaviors for dogs with otitis in the Midwestern United States.

Pseudomonas aeruginosa (P.aeruginosa) can cause otitis in dogs that is nonresponsive to empirical therapy. This study evaluated P.aeruginosa isolates (N=216) from canine ear swabs submitted to the Kansas State Veterinary Diagnostic Laboratory from 2018-2020 to create an antibiogram and minimum inhibitory concentration distributions using Clinical Laboratory Standards Institutes breakpoints. Multidrug resistance was defined as non-susceptibility to ≥1 drug from ≥3 antimicrobial classes. Submitting veterinarians (N=83) were invited to complete a survey about antimicrobial use and otitis management. Susceptibility was higher for aminoglycosides [gentamicin (82%, 177/216) and amikacin (81%, 175/216)] than fluoroquinolones [marbofloxacin (67%, 145/216), enrofloxacin (32%, 70/216), and orbifloxacin (18%, 39/216)]. Most responding veterinarians (54%, 15/28) prescribe topical aminoglycosides as first-line therapy for canine otitis, but 71% (15/21) prescribe fluoroquinolones if rods are seen cytologically. Ceftazidime, imipenem, and piperacillin-tazobactam showed high susceptibility and are used rarely. Multidrug resistance was present in 13% (28/216) of isolates. Based on in vitro susceptibility, topical aminoglycosides might be more effective than fluoroquinolones for P.aeruginosa otitis, but efficacy studies are required. Susceptibility testing is encouraged for cases not responding to empirical therapy but has limitations because topical preparations have high concentrations and otic breakpoints are not available.

Significant increase in azithromycin “resistance” and susceptibility to ceftriaxone and cefixime in Neisseria gonorrhoeae isolates in 26 European countries, 2019

BackgroundThe European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) performs annual sentinel surveillance of Neisseria gonorrhoeae susceptibility to therapeutically relevant antimicrobials across the European Union/European Economic Area (EU/EEA). We present the Euro-GASP results from 2019 (26 countries), linked to patient epidemiological data, and compared with data from previous years.MethodsAgar dilution and minimum inhibitory concentration (MIC) gradient strip methodologies were used to determine the antimicrobial susceptibility (using EUCAST clinical breakpoints, where available) of 3239 N. gonorrhoeae isolates from 26 countries across the EU/EEA. Significance of differences compared with Euro-GASP results in previous years was analysed using Z-test and the Pearson's χ2 test was used to assess significance of odds ratios for associations between patient epidemiological data and antimicrobial resistance.ResultsEuropean N. gonorrhoeae isolates collected between 2016 and 2019 displayed shifting MIC distributions for; ceftriaxone, with highly susceptible isolates increasing over time and occasional resistant isolates each year; cefixime, with highly-susceptible isolates becoming increasingly common; azithromycin, with a shift away from lower MICs towards higher MICs above the EUCAST epidemiological cut-off (ECOFF); and ciprofloxacin which is displaying a similar shift in MICs as observed for azithromycin. In 2019, two isolates displayed ceftriaxone resistance, but both isolates had MICs below the azithromycin ECOFF. Cefixime resistance (0.8%) was associated with patient sex, with resistance higher in females compared with male heterosexuals and men-who-have-sex-with-men (MSM). The number of countries reporting isolates with azithromycin MICs above the ECOFF increased from 76.9% (20/26) in 2016 to 92.3% (24/26) in 2019. Isolates with azithromycin MICs above the ECOFF (9.0%) were associated with pharyngeal infection sites. Following multivariable analysis, ciprofloxacin resistance remained associated with isolates from MSM and heterosexual males compared with females, the absence of a concurrent chlamydial infection, pharyngeal infection sites and patients ≥ 25 years of age.ConclusionsResistance to ceftriaxone and cefixime remained uncommon in EU/EEA countries in 2019 with a significant decrease in cefixime resistance observed between 2016 and 2019. The significant increase in azithromycin “resistance” (azithromycin MICs above the ECOFF) threatens the effectiveness of the dual therapy (ceftriaxone + azithromycin), i.e., for ceftriaxone-resistant cases, currently recommended in many countries internationally and requires close monitoring.

Use of Novel Antibiograms to Determine the Need for Earlier Susceptibility Testing and Administration for New β-Lactam/β-Lactamase Inhibitors in the United States.

Antimicrobial resistance is a global public health threat, and gram-negative bacteria, such as Enterobacterales and Pseudomonas aeruginosa, are particularly problematic with difficult-to-treat resistance phenotypes. To reduce morbidity and mortality, a reduction in the time to effective antimicrobial therapy (TTET) is needed, especially among critically ill patients. The antibiogram is an effective clinical tool that can provide accurate antimicrobial susceptibility information and facilitate early antimicrobial optimization, decrease TTET, and improve outcomes such as mortality, hospital length of stay, and costs. Guidance is lacking on how to validate the susceptibility to new antibacterial agents. Commonly used traditional and combination antibiograms may not adequately assist clinicians in making treatment decisions. Challenges with the current susceptibility testing of new β-lactam/β-lactamase inhibitor combinations persist, impacting the appropriate antibacterial choice and patient outcomes. Novel antibiograms such as syndromic antibiograms that incorporate resistant gram-negative phenotypes and/or minimum inhibitory concentration distributions may assist in determining the need for earlier susceptibility testing or help define an earlier optimal use of the new β-lactam/β-lactamase inhibitors. The purpose of this review is to emphasize novel antibiogram approaches that are capable of improving the time to susceptibility testing and administration for new β-lactam/β-lactamase inhibitors so that they are earlier in a patient’s treatment course.

Minimum inhibitory concentration of cycloserine against Mycobacterium tuberculosis using the MGIT 960 system and a proposed critical concentration

We aimed to determine the breakpoint of cycloserine (CS) susceptibility in MGIT and to describe the molecular characteristics of CS-resistant Mycobacterium tuberculosis (MTB) isolates. A total of 124 MTB isolates were recruited in our analysis. Minimum inhibitory concentration (MIC) was determined using the MGIT system. The mutations of MTB isolates within alr, ddl, ald, and cycA, potentially conferring CS resistance were analyzed by the whole-genome sequencing. In vitro drug susceptibility testing of isolates with doubling concentrations of CS revealed that the modal MIC values was 4 mg/L for MGIT, accounting for 35.5% (44/124) of isolates tested. Seven isolates harbored mutations conferring CS resistance, consisting of five with alr mutations and two with ald mutations. On the basis of the MIC distributions of wild-type and resistotype populations, we proposed a tentative epidemiologic cut-off value of 16 mg/l. The proportion of CS resistance in extensively drug-resistant TB was significantly higher than that of multidrug-resistant TB. In conclusion, we propose critical concentration for MGIT 960 to properly diagnose CS-resistant MTB and demonstrate that mutations in alr and ald genes are the major mechanism conferring CS resistance in clinical isolates.

Establishment of Epidemiological Resistance Cut-Off Values of Aquatic Aeromonas to Eight Antimicrobial Agents.

The abuse of antibiotics in aquaculture has led to the increasing rate of antibiotic resistance of aquatic bacteria including Aeromonas, which is an increasing threat to environmental and human health. To date, no epidemiological cut-off values (COWT) for Aeromonas spp. have been established by the Clinical and Laboratory Standards Institute nor the European Commission on Antimicrobial Susceptibility Testing. In this study, commercially prepared minimum inhibitory concentration (MIC) test 96-well plates (dry-form plates) were used to determine the MIC of eight antimicrobial agents against 556 Aeromonas strains. The obtained MIC distributions were simulated and analyzed by NRI and ECOFFinder to obtain tentative COWT values for Aeromonas spp. The COWT values of eight kinds of representative antimicrobial agents including trimethoprim–sulfamethoxazole, erythromycin, doxycycline, neomycin, colistin, florfenicol, enrofloxacin, and ceftazidime for Aeromonas spp. were established and were 0.25, 64/32, 4/2, 8, 4, 1, 0.062/0.125, and 0.5 μg/mL, respectively. Results showed that Aeromonas spp. had a very high proportion of non-wild-type strains to enrofloxacin, florfenicol, and doxycycline, which are the most widely used antimicrobials in aquaculture. The COWT values for Aeromonas spp. obtained in this study can contribute to the final establishment of COWT for Aeromonas spp. internationally.

Epidemiological cut-off values for a 96-well broth microdilution plate for high-throughput research antibiotic susceptibility testing of M. tuberculosis.

Drug susceptibility testing of M. tuberculosis is rooted in a binary susceptible/resistant paradigm. While there are considerable advantages in measuring the minimum inhibitory concentrations (MICs) of a panel of drugs for an isolate, it is necessary to measure the epidemiological cut-off values (ECOFF/ECVs) to permit comparison with qualitative data. Here we present ECOFF/ECVs for 13 anti-tuberculosis compounds, including bedaquiline and delamanid, derived from 20 637 clinical isolates collected by 14 laboratories based in 11 countries on five continents. Each isolate was incubated for 14 days on a dry 96-well broth microdilution plate and then read. Resistance to most of the drugs due to prior exposure is expected and the MIC distributions for many of the compounds are complex, and therefore a phenotypically wild-type population could not be defined. Since a majority of samples also underwent genetic sequencing, we defined a genotypically wild-type population and measured the MIC of the 99th percentile by direct measurement and via fitting a Gaussian using interval regression. The proposed ECOFF/ECVs were then validated by comparing with the MIC distributions of high-confidence genetic variants that confer resistance and with qualitative drug susceptibility tests obtained via the Mycobacterial Growth Indicator Tube (MGIT) system or Microscopic-Observation Drug Susceptibility (MODS) assay. These ECOFF/ECVs will inform and encourage the more widespread adoption of broth microdilution: this is a cheap culture-based method that tests the susceptibility of 12–14 antibiotics on a single 96-well plate and so could help personalise the treatment of tuberculosis.