Corynebacterium diphtheriae and Corynebacterium ulcerans: development of EUCAST methods and generation of data on which to determine breakpoints.

BACKGROUND: Evidence-based clinical susceptibility breakpoints have been lacking for antimicrobial agents used for diphtheria. OBJECTIVES: We aimed to evaluate broth microdilution and disc diffusion methods and create a dataset of MIC values and inhibition zone diameters (ZDs) from which breakpoints could be determined. METHODS: We included 400 recent clinical isolates equally distributed by species (Corynebacterium diphtheriae and Corynebacterium ulcerans) and by national surveillance programmes (France and Germany). Non-duplicate toxigenic and non-toxigenic isolates were chosen to enable the inclusion of a diversity of susceptibility levels for the 13 agents tested. Broth microdilution and disc diffusion, using EUCAST methodology for fastidious organisms, were used. RESULTS: The distributions of MIC and ZD values were largely in agreement among methods and countries. Breakpoints to allow categorization of WT isolates as susceptible, i.e. susceptible (S) or susceptible, increased exposure (I) were determined for 12 agents. The data supported a breakpoint for benzylpenicillin and amoxicillin of resistant (R) > 1 mg/L since WT isolates were inhibited by 1 mg/L or less. WT isolates were categorized as I (S ≤ 0.001 mg/L) for benzylpenicillin, emphasizing the need for increased exposure, and S (S ≤ 1 mg/L) for amoxicillin. Erythromycin breakpoints were set at S ≤ 0.06 mg/L and R > 0.06 mg/L. The corresponding ZD breakpoints were determined for all agents except amoxicillin, for which categorization was based on benzylpenicillin results. CONCLUSIONS: This work provided a large set of antimicrobial susceptibility data for C. diphtheriae and C. ulcerans, using a harmonized methodology. The dataset allowed EUCAST and experts in the diphtheria field to develop evidence-based breakpoints in January 2023.

Cutibacterium avidum: A Potent and Underestimated Pathogen in Prosthetic Hip Joint Infections.

Cutibacterium avidum has recently been reported as a rare cause of prosthetic joint infections (PJIs), contrary to Cutibacterium acnes, which is well established as a cause of PJIs, especially in shoulder arthroplasties. Two specific risk factors for PJI due to C. avidum have been reported: obesity and the skin incision approach. Here, we report four cases of hip PJIs caused by C. avidum admitted over a 30-month period at a single center. Whole-genome sequencing revealed that the four C. avidum strains were all individual strains and did not originate from a common source, such as an outbreak. Antibiotic susceptibility tests showed that the isolates were fully susceptible, and none carried known antibiotic resistance genes. In conclusion, the occurrence of four cases of PJI caused by C. avidum over a limited time at a single center may indicate that this pathogen is underestimated and is either emerging or more common than previously recognized. The patients presented overt signs of infection during surgery, indicating that C. avidum is a virulent pathogen. None of the previously reported risk factors for C. avidum PJI applied to these patients as only one was obese and none were operated on using a direct anterior skin incision approach.

Vibrio species: development of EUCAST susceptibility testing methods and MIC and zone diameter distributions on which to determine clinical breakpoints.

OBJECTIVES: Most human infections caused by Vibrio spp. do not warrant antimicrobial treatment but in severe cases, targeted antimicrobial treatment can be lifesaving. For Vibrio spp., standardized antimicrobial susceptibility testing (AST) guidelines with EUCAST methodology are lacking. In this study, we aimed to produce data suitable for EUCAST to establish clinical MIC breakpoints and zone diameter correlates for Vibrio spp. METHODS: An intercontinental collection (N = 524) comprising five important Vibrio spp. (V. alginolyticus, V. cholerae, V. fluvialis, V. parahaemolyticus and V. vulnificus) was organized. All isolates were subjected to broth microdilution (BMD) against 11 antimicrobial agents according to ISO 20776-1 using unsupplemented Mueller-Hinton broth on freeze-dried Sensititre panels (Thermo Scientific, UK), and most isolates (n = 371) were also tested with disc diffusion according to EUCAST methodology for non-fastidious organisms. RESULTS: Aggregated results were used to generate MIC and zone diameter distributions and to prepare graphs of MIC-zone diameter correlation. Based on these results, the EUCAST Steering Committee determined clinical susceptible (S) and resistant (R) MIC (mg/L) breakpoints (S≤/R>) for the five Vibrio spp. for piperacillin/tazobactam (1/1), cefotaxime (0.25/0.25), ceftazidime (1/1), meropenem (0.5/0.5), ciprofloxacin (0.25/0.25), levofloxacin (0.25/0.25), azithromycin (4/4), doxycycline (0.5/0.5) and trimethoprim/sulfamethoxazole (0.25/0.25). The corresponding zone diameter breakpoints were identified. CONCLUSIONS: We demonstrated the validity of using standard BMD and EUCAST disc diffusion methodology for AST of five Vibrio spp., and generated suitable data to allow EUCAST to determine clinical MIC and zone diameter breakpoints for five pathogenic Vibrio spp., including both non-toxigenic and toxigenic V. cholerae.

Evaluation of prolonged incubation time of 16-20 h with the EUCAST rapid antimicrobial susceptibility disc diffusion testing method.

OBJECTIVES: Antimicrobial resistance rates are continuously increasing, driving the need for rapid antimicrobial susceptibility testing (RAST) results, especially in the treatment of bloodstream infections. The EUCAST RAST method performed directly from positive blood cultures with incubation times from 4 to 8 h was developed in 2018 and is now used in many laboratories. To increase the practicality of the method, an extended incubation time of 16 and 20 h was evaluated in this study. METHOD: Blood culture bottles were spiked with clinical isolates (n = 325) of the seven most important sepsis pathogens. The EUCAST RAST method was performed, extending the incubation time to 16 and 20 h. Broth microdilution (BMD) was used as a reference, except for screening tests where standard disc diffusion or presence of resistance genes was used. RESULTS: Inhibition zones were possible to read for all species-agent combinations. For 16 and 20 h, the MIC zone diameter correlations were sufficiently similar to allow establishment of common breakpoints for the time interval of 16-20 h. The proportion of isolates in the area of technical uncertainty was, on average, 6% for all species and the number of errors were low, with <1% false-resistant and <0.5% false-susceptible results. CONCLUSIONS: This study shows that, for EUCAST RAST, prolonging the recommended incubation to 16-20 h is possible and can be used as a complement when the intended shorter incubation is not possible to achieve. The introduction of the prolonged incubation will increase the usefulness of the EUCAST RAST method in clinical laboratories with limited opening hours.

Performance of automated antimicrobial susceptibility testing for the detection of antimicrobial resistance in gram-negative bacteria: a NordicAST study.

Automated testing of antimicrobial susceptibility is common in clinical microbiology laboratories but their ability to detect low-level resistance has been questioned. This Nordic multicentre study aimed to evaluate the performance of commercially available automated AST systems. A phenotypically well-characterised collection of gram-negative bacilli (Escherichia coli (n = 7), Klebsiella pneumoniae (n = 6) and Pseudomonas aeruginosa (n = 7)) with and without resistance mechanisms was examined by Danish (n = 1), Finnish (n = 6), Norwegian (n = 16) and Swedish (n = 5) laboratories. Minimum inhibitory concentrations (MICs) were determined for 12 antimicrobials with automated systems and compared with MICs obtained with gold standard broth microdilution. The automated systems used were VITEK 2 (n = 23), Phoenix (n = 4), MicroScan (n = 1), and ARIS (n = 1). Very major errors were identified for six antimicrobials; cefotaxime (6.9%), meropenem (0.4%), ciprofloxacin (0.7%), ertapenem (4.3%), amikacin (3.4%) and colistin (6.4%). Categorical agreement of MIC for the automated systems compared to broth microdilution ranged from 83% for imipenem to 100% for ampicillin and trimethoprim-sulfamethoxazole. The analysis revealed several important antimicrobials where resistance was underestimated, potentially with significant consequences in patient treatment. The results cast doubt on the use of automated AST in the management of patients with serious infections and suggests that more work is needed to define their limitations.

Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host.

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes a wide range of airway diseases. NTHi has a plethora of mechanisms to colonize while evading the host immune system for the establishment of infection. We previously showed that the outer membrane protein P5 contributes to bacterial serum resistance by the recruitment of complement regulators. Here, we report a novel role of P5 in maintaining bacterial outer membrane (OM) integrity and protein composition important for NTHi-host interactions. In silico analysis revealed a peptidoglycan-binding motif at the periplasmic C-terminal domain (CTD) of P5. In a peptidoglycan-binding assay, the CTD of P5 (P5CTD) formed a complex with peptidoglycan. Protein profiling analysis revealed that deletion of CTD or the entire P5 changed the membrane protein composition of the strains NTHi 3655Δp5CTD and NTHi 3655Δp5, respectively. Relative abundance of several membrane-associated virulence factors that are crucial for adherence to the airway mucosa, and serum resistance were altered. This was also supported by similar attenuated pathogenic phenotypes observed in both NTHi 3655Δp5 CTD and NTHi 3655Δp5. We found (i) a decreased adherence to airway epithelial cells and fibronectin, (ii) increased complement-mediated killing, and (iii) increased sensitivity to the ß-lactam antibiotics in both mutants compared to NTHi 3655 wild-type. These mutants were also more sensitive to lysis at hyperosmotic conditions and hypervesiculated compared to the parent wild-type bacteria. In conclusion, our results suggest that P5 is important for bacterial OM stability, which ultimately affects the membrane proteome and NTHi pathogenesis.

Assessing the quality of the anaerobic environment - a method developed to support EUCAST disk diffusion of anaerobic bacteria.

The purpose of this study is to establish a method for assessing the anaerobic environment for EUCAST disk diffusion antimicrobial susceptibility testing (AST) of anaerobic bacteria on fastidious anaerobe agar with 5% mechanically defibrinated horse blood (FAA-HB). The method utilizes the association between a decrease in the metronidazole disk zone diameter and increasing oxygen levels with an aerotolerant Clostridium perfringens strain DSM 25589 (CCUG 75076 and NCTC 14679). The C. perfringens strain was tested on FAA-HB with a McFarland 1 inoculum and a metronidazole 5 µg disk. FAA-HB was incubated for 16-20 h at 35-37°C. The association between oxygen levels (0, 0.16, 1, 2, and 4% oxygen) and the metronidazole zone diameter was determined. Reproducibility at 0% oxygen was investigated as part of a European multi-centre study of disk diffusion of anaerobic bacteria. The median zone diameters (n=12) at each oxygen level were 29 mm (0%), 21 mm (0.16%), 16 mm (1%), 15 mm (2%), and 15 mm (4%). The metronidazole zone diameters at 0% oxygen from the multi-centre reproducibility-study had a median of 29 mm and a 95%-percentile range of 25-33 mm (n=236). Only one reading was below 25 mm. Based on our results, a zone diameter of ≥25 mm using a metronidazole 5 µg disk and the C. perfringens strain, tested with EUCAST recommendations, can be used to indicate that the anaerobic environment is of sufficient quality for culture and disk diffusion AST. EUCAST has included the method as part of the quality control for AST of anaerobic bacteria.

Microbiological Epidemiology of Invasive Infections Due to Non-Beta-Hemolytic Streptococci, France, 2021.

Non-beta-hemolytic streptococci (NBHS), also referred to as viridans streptococci, represent an underestimated cause of human invasive diseases. Their resistance to antibiotics, including beta-lactam agents, often complicate their therapeutic management. A prospective multicenter study was conducted by the French National Reference Center for Streptococci between March and April 2021 to describe the clinical and microbiological epidemiology of invasive infections due to NBHS, excluding pneumococcus. A total of 522 NBHS invasive cases were collected. Distribution among streptococcal groups was: Streptococcus anginosus (33%), Streptococcus mitis (28%), Streptococcus sanguinis (16%), Streptococcus bovis/equinus (15%), Streptococcus salivarius (8%), and Streptococcus mutans (<1%). Median age of infection was 68 years old (range <1 day to 100 years). Cases were more frequent in male patients (gender ratio M/F 2.1:1) and manifested mainly as bacteremia without focus (46%), intra-abdominal infections (18%) and endocarditis (11%). All isolates were susceptible to glycopeptides and displayed low-level inherent gentamicin resistance. All isolates of the S. bovis/equinus, S. anginosus, and S. mutans groups were susceptible to beta-lactams. Conversely, nonsusceptibility to beta-lactams was found in 31%, 28%, and 52% of S. mitis, S. salivarius, and S. sanguinis isolates, respectively. The screening for beta-lactam resistance using the recommended one unit benzylpenicillin disk screening failed to detect 21% of resistant isolates (21/99). Last, overall resistance rates to the alternative anti-streptococcal molecules clindamycin and moxifloxacin were 29% (149/522) and 1.6% (8/505), respectively. IMPORTANCE NBHS are recognized as opportunistic pathogens particularly involved in infections of the elderly and immunocompromised patients. This study underlines their importance as common causes of severe and difficult-to-treat infections such as endocarditis. Although species of the S. anginosus and S. bovis/equinus groups remain constantly susceptible to beta-lams, resistance in oral streptococci exceeds 30% and screening techniques are not fully reliable. Therefore, accurate species identification and antimicrobial susceptibility testing by MICs determination appears essential for the treatment of NBHS invasive infections, together with continued epidemiological surveillance.

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 <28 mm in B. fragilis indicated presence of cfiA. Piperacillin-tazobactam had the most false susceptible results. Categorical errors for this antimicrobial were particularly prevalent in cfiA-positive strains, and piperacillin-tazobactam had the highest number of comments describing zone reading difficulties. 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.

Contemporary Considerations for Establishing Reference Methods for Antibacterial Susceptibility Testing.

Antibacterial susceptibility testing (AST) is performed to guide therapy, perform resistance surveillance studies, and support development of new antibacterial agents. For 5 decades, broth microdilution (BMD) has served as the reference method to assess in vitro activity of antibacterial agents against which both novel agents and diagnostic tests have been measured. BMD relies on in vitro inhibition or killing of bacteria. It is associated with several limitations: it is a poor mimic of the in vivo milieu of bacterial infections, requires multiple days to perform, and is associated with subtle, difficult to control variability. In addition, new reference methods will soon be needed for novel agents whose activity cannot be evaluated by BMD (e.g., those that target virulence). Any new reference methods must be standardized, correlated with clinical efficacy and be recognized internationally by researchers, industry, and regulators. Herein, we describe current reference methods for in vitro assessment of antibacterial activity and highlight key considerations for the generation of novel reference methods.

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

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

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

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.

Evaluation of ten brands of pre-poured Mueller-Hinton agar plates for EUCAST disc diffusion testing.

OBJECTIVES: Mueller-Hinton agar (MHA) is recommended by EUCAST and CLSI for disc diffusion antimicrobial susceptibility testing (AST). We have previously investigated the quality of dehydrated MHA from several manufacturers. In this study, we evaluated the performance of ten commercial brands of pre-poured MHA plates. METHODS: AST was performed according to EUCAST methodology and results analyzed against targets and ranges in EUCAST quality control (QC) tables. MHA plates from different brands were tested in triplicate against four non-fastidious QC strains. The agar depth and pH were measured for all products. RESULTS: The best performance was observed for MHA from Becton Dickinson (BBL MHA II), bioMérieux (MHE agar) and Hardy Diagnostics, for which >97% of zone diameters were within QC ranges and >60% on target ±1 mm. The poorest performance was seen for plates from HiMedia (MHA and MHA no. 2), where 20% and 18% of readings were outside the QC ranges, respectively. The differences in pH and agar depth of the products were small and mostly within EUCAST specifications. DISCUSSION: The accuracy and reproducibility of disc diffusion AST depends on standardised procedures and high-quality discs and media. The performance among ten brands of pre-poured MHA plates differed significantly. The results indicate a poorer performance for pre-poured commercial plates as compared to in-house prepared plates from dehydrated powder of corresponding brands in our previous study. Manufacturers and clinical laboratories have a shared responsibility for the quality of AST. EUCAST provides QC criteria to be used both by manufacturers and laboratories.

Cefiderocol: EUCAST criteria for disc diffusion and broth microdilution for antimicrobial susceptibility testing.

OBJECTIVES: The reproducibility of cefiderocol MIC determination using broth microdilution (BMD) in iron-depleted CAMHB (ID-CAMHB) was investigated, and the EUCAST disc diffusion (DD) method for cefiderocol susceptibility testing was developed and validated against reference BMD. METHODS: Cefiderocol values were determined for wild-type (WT) and non-WT isolates using BMD plates with ID-CAMHB (Thermo Scientific, Oakwood, USA) per EUCAST guidelines. DD was performed using standard EUCAST methodology on unsupplemented Mueller-Hinton agar with cefiderocol 30 µg discs. Control agents were included in all tests. MICs were correlated with zone diameters (ZD), and ZD breakpoints (BP) best corresponding to the MIC BPs were determined. Areas of technical uncertainty (ATU) were included where appropriate. External laboratory validation of cefiderocol DD was performed per the EUCAST SOP 9.2. RESULTS: MIC and ZD distributions for cefiderocol against WT isolates were established. Cefiderocol ZD BPs were set at susceptible ≥22 mm, resistant <22 mm for Enterobacterales and Pseudomonas aeruginosa and ATUs were decided. For Acinetobacter baumannii and Stenotrophomonas maltophilia, ZD cut-off values of ≥17 mm and ≥20 mm corresponded to MIC values of ≤2 and ≤0.5 mg/L, respectively. Cefiderocol ZDs for Escherichia coli ATCC 25922 (target 27 mm) and P. aeruginosa ATCC 27853 (target 26 mm) were within ±3 mm of the target values. For DD, there was no problematic variation between discs, media or laboratories. CONCLUSIONS: DD is a robust and easy-to-perform method for cefiderocol susceptibility testing. For isolates with results in the ATU, an MIC test should be performed to confirm the results.

Revisiting colistin susceptibility testing: will adding calcium to Mueller-Hinton agar improve the detection of colistin resistance?

OBJECTIVE: The aim was to investigate whether adding calcium to Mueller-Hinton agar for gradient MIC or disc diffusion tests could improve separation between colistin-susceptible and -resistant populations of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. and if this method could provide a reliable screening test for colistin resistance in routine laboratories. METHODS: An isolate collection of 57 E. coli, K. pneumoniae, P. aeruginosa and Acinetobacter spp. was tested. Ca2+ in concentrations from 2.5 to 40 mM was added to the Mueller-Hinton agar plates used for gradient MIC and disc diffusion tests. Broth microdilution (ISO 20776-1) MIC determination was used as reference. Escherichia coli and K. pneumoniae were investigated for colistin resistance genes. RESULTS: Results were similar for gradient tests and disc diffusion for all species. Correlation between phenotypic expression of resistance and resistance genes was not absolute. Addition of Ca2+ to Mueller-Hinton agar improved separation between colistin-susceptible and -resistant isolates for E. coli. For K. pneumoniae, separation was improved for isolates with mcr genes, but not for isolates harbouring other colistin resistance mechanisms. To further increase the concentrations of Ca2+ did not improve the separation between susceptible and resistant isolates of E. coli and K. pneumoniae. For P. aeruginosa and Acinetobacter species, addition of Ca2+ did not improve separation between susceptible and resistant populations. DISCUSSION: The results from this study show that addition of Ca2+ to the Mueller-Hinton agar does not sufficiently improve detection of colistin resistance by gradient MIC or disc diffusion tests for use in a routine laboratory.

Development of a EUCAST disk diffusion method for the susceptibility testing of rapidly growing anaerobic bacteria using Fastidious Anaerobe Agar (FAA): a development study using Bacteroides species.

OBJECTIVES: Antimicrobial resistance among anaerobic bacteria is increasing, leading to a growing demand for inexpensive and reliable susceptibility testing methods. The aim of this study was to determine the suitability of Fastidious Anaerobe Agar (FAA) as a medium for disk diffusion for rapidly growing anaerobic bacteria. METHODS: Reproducibility of zone diameters and quality of growth were tested using six quality control (QC) strains. We compared four anaerobic incubation systems, two incubation temperatures (35°C and 37°C), and FAA from four manufacturers. The effect of incubation for 16-20 hours instead of 24 hours was tested on ten randomly selected isolates of the Bacteroides fragilis group. The final method was tested on 170 clinical B. fragilis-group isolates and compared to agar dilution MICs. RESULTS: After 24 hours' incubation, all QC strains demonstrated confluent growth. The different anaerobic incubation systems were equal regarding quality of growth and zone diameters. Incubation at 35°C resulted in slightly larger zones (1-2 mm) than at 37°C. Except for Acumedia FAA, the different manufacturers showed good agreement in zone diameters. All B. fragilis-group isolates displayed confluent growth after 16-20 hours. Metronidazole inhibition zones correlated well with the reference MICs. There was an area of poorer separation for meropenem and piperacillin-tazobactam between 19-27 and 14-23 mm respectively. Prolonged incubation (40-44 h) of clindamycin resulted in better separation and the area of overlap was reduced from 13 to 8 mm compared with 16-20 hours' incubation. CONCLUSION: FAA is a suitable medium for disk diffusion of these rapidly growing anaerobic bacteria.

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.

EUCAST rapid antimicrobial susceptibility testing (RAST) in blood cultures: validation in 55 European laboratories.

OBJECTIVES: When bloodstream infections are caused by resistant bacteria, rapid antimicrobial susceptibility testing (RAST) is important for adjustment of therapy. The EUCAST RAST method, directly from positive blood cultures, was validated in a multi-laboratory study in Europe. METHODS: RAST was performed in 40 laboratories in northern Europe (NE) and 15 in southern Europe (SE) from clinical blood cultures positive for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus or Streptococcus pneumoniae. Categorical results at 4, 6 and 8 h of incubation were compared with results for EUCAST standard 16-20 h disc diffusion. The method, preliminary breakpoints and the performance of the laboratories were evaluated. RESULTS: The total number of isolates was 833/318 in NE/SE. The number of zone diameters that could be read (88%, 96% and 99%) and interpreted (70%, 81% and 85%) increased with incubation time (4, 6 and 8 h). The categorical agreement was acceptable, with total error rates in NE/SE of 2.4%/4.9% at 4 h, 1.1%/3.5% at 6 h and 1.1%/3.3% at 8 h. False susceptibility at 4, 6 and 8 h of incubation was below 0.3% and 1.1% in NE and SE, respectively, and the corresponding percentages for false resistance were below 1.9% and 2.8%. After fine-tuning breakpoints, more zones could be interpreted (73%, 89% and 93%), with only marginally affected error rates. CONCLUSIONS: The EUCAST RAST method can be implemented in routine laboratories without major investments. It provides reliable antimicrobial susceptibility testing results for relevant bloodstream infection pathogens after 4-6 h of incubation.

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 the Mycobacterium tuberculosis complex (MTBC). The variability in the results obtained with these methods hampers setting 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 a standardized reference method for MIC determination for MTBC. METHODS: The EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared protocols of MIC determination with regard to medium, inoculum preparation, antituberculous agent preparation, incubation, reading of the results and interpretation. RECOMMENDATIONS: The EUCAST reference method of MIC determination for MTBC is the broth microdilution method in Middlebrook 7H9-10% OADC medium. The final inoculum is a 105 CFU/mL suspension, obtained from a 10-2 dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonies with 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 an inverted mirror as soon as the 1:100 diluted control (i.e. 103 CFU/mL suspension) shows visual growth. The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth. Mycobacterium tuberculosis H37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within the range 0.03-0.12 for isoniazid, 0.12-0.5 for levofloxacin and 0.25-1 mg/L for amikacin. CONCLUSIONS: The EUCAST reference method for MTBC was endorsed by EUCAST after public consultation and will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference method is not primarily intended to be used under routine conditions and the AST methods will need to be calibrated against this reference method to be used with EUCAST breakpoints.