Antimicrobial susceptibility testing of Bacteroides species by disk diffusion: The NordicAST Bacteroides study

Objectives - Antimicrobial susceptibility testing (AST) of anaerobic bacteria has until recently been done by MIC methods. We have carried out a multi-centre evaluation of the newly validated EUCAST disk diffusion method for AST of Bacteroides spp. Methods - A panel of 30 Bacteroides strains was assembled based on reference agar dilution MICs, resistance gene detection and quantification of cfiA carbapenemase gene expression. Nordic clinical microbiology laboratories (n = 45) performed disk diffusion on Fastidious Anaerobe Agar with 5% mechanically defibrinated horse blood (FAA-HB) for piperacillin-tazobactam, meropenem and metronidazole. Results - A total of 43/45 (95.6%) laboratories carried out disk diffusion per protocol. Intraclass correlation coefficients were 0.87 (0.80–0.93) for piperacillin-tazobactam, 0.95 (0.91–0.97) for meropenem and 0.89 (0.83–0.94) for metronidazole. For metronidazole, one media lot yielded smaller zones and higher variability than another. Piperacillin-tazobactam and meropenem zone diameters correlated negatively with cfiA expression. A meropenem zone diameter of Conclusions - Inter-laboratory agreement by disk diffusion was good or very good. The main challenges were media-related variability for metronidazole and categorical disagreement with the reference method for piperacillin-tazobactam in some cfiA-positive strains. An area of technical uncertainty specific for such strains may be warranted

Multicentre testing of the EUCAST broth microdilution reference method for MIC determination on mycobacterium tuberculosis

Objectives: the first objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for antimycobacterial susceptibility testing (AMST), launched in 2016, was to set a reference method for determining the MICs of antituberculous agents, since many protocols are used worldwide and a consensus one is needed for the determination of microbiological breakpoints. Methods: during 2017 and 2018, MIC determination protocols were evaluated prospectively in a multicentre study within the four AMST laboratories. MIC results were obtained for isoniazid, levofloxacin and amikacin on the reference strain Mycobacterium tuberculosis H37Rv ATCC 27294. Broth microdilution (BMD) in Middlebrook 7H9 and solid medium dilution (SMD) in Middlebrook 7H10 were performed using two inoculum concentrations. MICs were interpreted with regard to visual and 99% inhibition after 7, 14 or 21 days of incubation for BMD and 21 days for SMD. Results: following the EUCAST reference protocol, intra- and inter-assay agreements were within ±1 MIC dilution for >95% of the observations for the three drugs in both methods. MIC values, presented as MIC mode (range) for BMD and SMD respectively, were: 0.03 (0.015-0.06) mg/L and 0.12 (0.06-0.25) mg/L for isoniazid, 0.25 mg/L (0.25-0.5) and 0.5 mg/L (0.12-0.5) for levofloxacin, and 0.5 mg/L (0.5-1.0) and 0.5 mg/L (0.5-1.0) for amikacin. Conclusions: both SMD and BMD were reproducible and eligible as a reference method for MIC determination of the Mycobacterium tuberculosis complex (MTBC). BMD was finally selected as the EUCAST reference method. From now on it will be used to set epidemiological cut-off values and clinical breakpoints of new and old antituberculous agents

Antimicrobial susceptibility testing of mycobacterium tuberculosis complex isolates - the EUCAST broth microdilution reference method for MIC determination

Scope:Several methods are used worldwide for antibiotic susceptibility testing (AST) for theMycobac-terium tuberculosiscomplex (MTBC). The variability in the results obtained with these methods hamperssetting epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines.Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MICs)need to be standardized for MTBC isolates for old and new agents. Our objective was to establish astandardized reference method for MIC determination for MTBC.Methods:The EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared pro-tocols of MIC determination with regard to medium, inoculum preparation, antituberculous agentpreparation, incubation, reading of the results and interpretation.Recommendations:The EUCAST reference method of MIC determination for MTBC is the broth micro-dilution method in Middlebrook 7H9-10% OADC medium. Thefinal inoculum is a 105CFU/mL suspension,obtained from a 10 2dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonieswith glass beads before suspending them in sterile water. The culture is maintained in a U-shaped 96-well polystyrene microtitre sterile plate with a lid incubated at 36 ±1 C. Reading is done using aninverted mirror as soon as the 1:100 diluted control (i.e. 103CFU/mL suspension) shows visual growth.The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth.MycobacteriumtuberculosisH37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within therange 0.03e0.12 for isoniazid, 0.12e0.5 for levofloxacin and 0.25e1 mg/L for amikacin.Conclusions:The EUCAST reference method for MTBC was endorsed by EUCAST after public consultationand will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference methodis not primarily intended to be used under routine conditions and the AST methods will need to b

Performance of the EUCAST disc diffusion method and two MIC methods in detection of Enterobacteriaceae with reduced susceptibility to meropenem: the NordicAST CPE study.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadTo examine performance of EUCAST disc diffusion and supplementary MIC methods for detection of Enterobacteriaceae with reduced susceptibility to meropenem using EUCAST screening recommendations. Sixty-one Nordic laboratories delivered data on EUCAST disc diffusion (n = 61), semi-automated meropenem MIC (n = 23; VITEK2, n = 20 and Phoenix, n = 3) and gradient meropenem MIC (n = 58) methods. The strains (n = 27) included the major carbapenemase classes (A, n = 4; B, n = 9; D, n = 6) involved in the global spread of carbapenemase-producing Enterobacteriaceae (CPE) and non-CPE strains (n = 8) covering a range of broth microdilution (BMD) meropenem MICs. A triplicate Klebsiella variicola (meropenem MIC 0.5 mg/L) harbouring OXA-48 and Escherichia coli ATCC 25922 showed an overall good precision. Meropenem zone diameters below the EUCAST screening cut-off (<27 mm) were reported for strains with MIC ≥1 mg/L (n = 21), irrespective of resistance mechanism. For three strains (MIC = 0.5 mg/L) with OXA-48/-181, eight laboratories provided meropenem zone diameters above the screening cut-off. Very major errors (VMEs) were not observed. The overall distributions of major errors (MEs) and minor errors (mEs) were 9% and 36% (disc diffusion), 26% and 18% (VITEK2) and 7% and 20% (gradient MIC), respectively. Differences in ME and mE distributions between disc diffusion and MIC gradient tests compared with semi-automated methods were significant (P < 0.0001), using BMD MICs as a reference for categorization. The EUCAST disc diffusion method is a robust method to screen for CPE but isolates with meropenem MICs <1 mg/L pose challenges. The high ME rate in semi-automated methods might deter appropriate use of carbapenems in CPE infections with limited therapeutic options.Northern Norway Regional Health Authority (Helse Nord RHF) Norwegian Directorate of Healt

Adaptation of Brucella melitensis Antimicrobial Susceptibility Testing to the ISO 20776 Standard and Validation of the Method

This article belongs to the Special Issue Emerging Themes in Brucella and Brucellosis.Brucellosis, mainly caused by Brucella (B.) melitensis, is associated with a risk of chronification and relapses. Antimicrobial susceptibility testing (AST) standards for B. melitensis are not available, and the agent is not yet listed in the EUCAST breakpoint tables. CLSI recommendations for B. melitensis exist, but they do not fulfill the requirements of the ISO 20776 standard regarding the culture medium and the incubation conditions. Under the third EU Health Programme, laboratories specializing in the diagnostics of highly pathogenic bacteria in their respective countries formed a working group within a Joint Action aiming to develop a suitable method for the AST of B. melitensis. Under the supervision of EUCAST representatives, this working group adapted the CLSI M45 document to the ISO 20776 standard after testing and validation. These adaptations included the comparison of various culture media, culture conditions and AST methods. A Standard Operation Procedure was derived and an interlaboratory validation was performed in order to evaluate the method. The results showed pros and cons for both of the two methods but also indicate that it is not necessary to abandon Mueller–Hinton without additives for the AST of B. melitensis.This research was funded by the EU Health Programme 2014–2020, through the Consumers, Health, Agriculture and Food Executive Agency (CHAFEA, European Commission), the Joint Action EMERGE (CHAFEA n° 677 066) and the Joint Action SHARP (848096-SHARP JA).info:eu-repo/semantics/publishedVersio

In Vitro Availability of Iron in High-Tannin Sorghum. Effect of Enzymatic Oxidation of Phenolic Compounds

Iron deficiency anemia is the most common nutritional disorder in the world. In low income countries, it is to a large extent caused by low bioavailability of iron in the vegetable diet. Vegetable diets contain large amounts of compounds that inhibit iron absorption, e.g. phytate and polyphenols (tannins). Polyphenols inhibit iron absorption by forming insoluble complexes with iron in the gastrointestinal tract. The aim of the present thesis was to investigate how enzymatic oxidation affects phenolic compounds and subsequently the bioavailability of iron in high-tannin cereals. The bioavailability of iron was estimated with an in vitro digestion method that has been validated against absorption data and found to be a suitable method. Oxidation of model phenolic compounds, i.e. tannic acid and green tea extract, with commercial polyphenol oxidase (PPO) significantly increased in vitro iron availability in a model food system. These results imply that the oxidation products have a lower iron binding capacity than the original phenolic compounds. Incubation of phytate-reduced flour of sorghum or millet also resulted in lower amounts of phenolic compounds and a higher availability of iron. The phenolic content in sorghum decreased by more than 50% with PPO incubation, and in vitro available iron increased by almost 100%. Furthermore, combining the enzymatic treatment with traditional processing methods, such as cooking, soaking, germination and lactic acid fermentation, resulted in a further improvement in iron availability. Polyphenol oxidase can be found in several common fruits and vegetables, and incubation of dephytinized high-tannin sorghum with dialyzed extract of pear, banana or avocado effectively increased the in vitro iron availability. The effect was more pronounced than with commercial PPO, and incubation with avocado extract improved the availability more than 1.7-fold. It is probable that peroxidase activity and organic acids in the fruit extracts contributed to the improved iron availability. Investigations of the effect of oxidation on phenolic structures, both simple phenolic compounds and complex structures such as sorghum tannins, showed a decrease after incubation with PPO. The decrease in tannins was probably due to coupled oxidation reactions involving simple phenols. Catechol (ortho-dihydroxyl) and galloyl (trihydroxyl) groups are identified as iron binding phenolic groups in plant foods. Model phenols containing catechol groups were more easily oxidized than those containing galloyl groups, while the percentage decrease in catechol and galloyl groups in sorghum phenols were similar. In combination with methods that reduce phytate, enzymatic oxidation of phenolic compounds may be a promising way to increase the bioavailability of iron in high-tannin cereals at the household level

In Vitro Availability of Iron in High-Tannin Sorghum. Effect of Enzymatic Oxidation of Phenolic Compounds

Iron deficiency anemia is the most common nutritional disorder in the world. In low income countries, it is to a large extent caused by low bioavailability of iron in the vegetable diet. Vegetable diets contain large amounts of compounds that inhibit iron absorption, e.g. phytate and polyphenols (tannins). Polyphenols inhibit iron absorption by forming insoluble complexes with iron in the gastrointestinal tract. The aim of the present thesis was to investigate how enzymatic oxidation affects phenolic compounds and subsequently the bioavailability of iron in high-tannin cereals. The bioavailability of iron was estimated with an in vitro digestion method that has been validated against absorption data and found to be a suitable method. Oxidation of model phenolic compounds, i.e. tannic acid and green tea extract, with commercial polyphenol oxidase (PPO) significantly increased in vitro iron availability in a model food system. These results imply that the oxidation products have a lower iron binding capacity than the original phenolic compounds. Incubation of phytate-reduced flour of sorghum or millet also resulted in lower amounts of phenolic compounds and a higher availability of iron. The phenolic content in sorghum decreased by more than 50% with PPO incubation, and in vitro available iron increased by almost 100%. Furthermore, combining the enzymatic treatment with traditional processing methods, such as cooking, soaking, germination and lactic acid fermentation, resulted in a further improvement in iron availability. Polyphenol oxidase can be found in several common fruits and vegetables, and incubation of dephytinized high-tannin sorghum with dialyzed extract of pear, banana or avocado effectively increased the in vitro iron availability. The effect was more pronounced than with commercial PPO, and incubation with avocado extract improved the availability more than 1.7-fold. It is probable that peroxidase activity and organic acids in the fruit extracts contributed to the improved iron availability. Investigations of the effect of oxidation on phenolic structures, both simple phenolic compounds and complex structures such as sorghum tannins, showed a decrease after incubation with PPO. The decrease in tannins was probably due to coupled oxidation reactions involving simple phenols. Catechol (ortho-dihydroxyl) and galloyl (trihydroxyl) groups are identified as iron binding phenolic groups in plant foods. Model phenols containing catechol groups were more easily oxidized than those containing galloyl groups, while the percentage decrease in catechol and galloyl groups in sorghum phenols were similar. In combination with methods that reduce phytate, enzymatic oxidation of phenolic compounds may be a promising way to increase the bioavailability of iron in high-tannin cereals at the household level