Burkholderia pseudomallei multi-centre study to establish EUCAST MIC and zone diameter distributions and epidemiological cut-off values.

OBJECTIVES: Melioidosis, caused by Burkholderia pseudomallei, requires intensive antimicrobial treatment. However, standardized antimicrobial susceptibility testing (AST) methodology based on modern principles for determining breakpoints and ascertaining performance of methods are lacking for B. pseudomallei. This study aimed to establish MIC and zone diameter distributions on which to set epidemiological cut-off (ECOFF) values for B. pseudomallei using standard EUCAST methodology for non-fastidious organisms. METHODS: Non-consecutive, non-duplicate clinical B. pseudomallei isolates (9-70 per centre) were tested at eight study centres against eight antimicrobials by broth microdilution (BMD) and the EUCAST disc diffusion method. Isolates without and with suspected resistance mechanisms were deliberately selected. The EUCAST Development Laboratory ensured the quality of study materials, and provided guidance on performance of the tests and interpretation of results. Aggregated results were analysed according to EUCAST recommendations to determine ECOFFs. RESULTS: MIC and zone diameter distributions were generated using BMD and disc diffusion results obtained for 361 B. pseudomallei isolates. MIC and zone diameter ECOFFs (mg/L; mm) were determined for amoxicillin-clavulanic acid (8; 22), ceftazidime (8; 22), imipenem (2; 29), meropenem (2; 26), doxycycline (2; none), tetracycline (8; 23), chloramphenicol (8; 22) and trimethoprim-sulfamethoxazole (4; 28). CONCLUSIONS: We have validated the use of standard BMD and disc diffusion methodology for AST of B. pseudomallei. The MIC and zone diameter distributions generated in this study allowed us to establish MIC and zone diameter ECOFFs for the antimicrobials studied. These ECOFFs served as background data for EUCAST to set clinical MIC and zone diameter breakpoints for B. pseudomallei.

EUCAST evaluation of 21 brands of Mueller-Hinton dehydrated media for disc diffusion testing.

OBJECTIVES: Mueller-Hinton (MH) agar is recommended by EUCAST and CLSI for disc diffusion antimicrobial susceptibility testing. Using EUCAST methodology, we evaluated the performance of 21 internationally available brands of dehydrated MH agar from 17 manufacturers. METHODS: MH plates were prepared in-house and evaluated against four quality control (QC) strains tested in triplicate, using EUCAST disc diffusion methodology. This resulted in 30 disc-QC strain combinations and 90 readings per MH brand. All brands were tested blindly and in parallel. Results were evaluated against targets and ranges in the EUCAST QC tables. The agar depth, pH and concentration of five cations were measured for all brands. RESULTS: Six brands of MH agar (Bio-Rad, Biolife, Oxoid, Sigma MH 2, BD BBL MH II and CRITERION) demonstrated excellent performance, with ≥99% of zone diameter readings within QC ranges and ≥70% on target ±1 mm. The poorest performance was seen for Biolab and Merck MH, with 10% (9/90) and 23% (21/90) of readings outside the QC ranges, respectively. Of all readings, 4.9% (93/1890) were out of range, mainly related to trimethoprim sulfamethoxazole (n = 25), aminoglycosides (n = 25) and fluoroquinolones (n = 15). The cation content differed considerably between the agars, and for four brands pH values were outside the acceptable range 7.2-7.4. DISCUSSIONS: This study evaluated the performance and content of 21 brands of MH dehydrated media. Six brands showed excellent performance with all investigated antimicrobial classes. Others exhibited problems with one or more classes of agents. This could partly be explained by differences in concentration of specific chemical components and pH.

EUCAST disc diffusion criteria for the detection of mecA-Mediated ß-lactam resistance in Staphylococcus pseudintermedius: oxacillin versus cefoxitin.

OBJECTIVES: Until recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommended the cefoxitin disc to screen for mecA-mediated ß-lactam resistance in Staphylococcus pseudintermedius. A recent study indicated that cefoxitin was inferior to oxacillin in this respect. We have re-evaluated cefoxitin and oxacillin discs for screening for methicillin resistance in S. pseudintermedius. METHODS: We included 224 animal and human S. pseudintermedius isolates from Europe (n = 108) and North America (n = 116), of which 109 were mecA-positive. Disc diffusion was performed per EUCAST recommendations using 30-µg cefoxitin and 1-µg oxacillin discs from three manufacturers and Mueller-Hinton agar from two manufacturers. RESULTS: Cefoxitin inhibition zones ranged from 6 to 33 mm for mecA-positive S. pseudintermedius (MRSP) and from 29 to 41 mm for mecA-negative S. pseudintermedius (MSSP). The corresponding oxacillin zone intervals were 6-20 mm and 19-30 mm. For cefoxitin 16% (95% CI 14.8-18.0%) of the isolates were in the area where positive and negative results overlapped. For oxacillin the corresponding number was 2% (1.6-2.9%). For oxacillin a breakpoint of susceptible (S) ≥ 20 mm and resistant (R) <20 mm resulted in only 0.4% and 1.1% very major error and major error rates respectively. CONCLUSIONS: This investigation confirms that the 1-µg oxacillin disc predicts mecA-mediated methicillin resistance in S. pseudintermedius better than the 30-µg cefoxitin disc. For a 1-µg oxacillin disc we propose that 20 mm should be used as cut off for resistance, i.e. isolates with a zone diameter <20 mm are resistant to all ß-lactam antibiotics except those with activity against methicillin-resistant staphylococci.

The quality of antimicrobial discs from nine manufacturers-EUCAST evaluations in 2014 and 2017.

OBJECTIVES: Antimicrobial discs for susceptibility testing can be obtained from many manufacturers. We evaluated the quality of discs from nine manufacturers in 2014 and 2017. METHODS: Antimicrobial discs of 16 agents from nine manufacturers were evaluated using EUCAST criteria. Discs were tested in triplicate on Müller-Hinton medium against EUCAST quality control (QC) strains. Mean values were compared with targets and ranges in the EUCAST QC tables. RESULTS: Three manufacturers (Becton Dickinson, Mast and Oxoid) demonstrated excellent and consistent disc quality both in 2014 and 2017. Manufacturers with discs of inadequate quality improved their results between the two periods. Overall, 92% (795/861) versus 97% (1038/1071) of zone diameter readings were within QC ranges and 58% (497/861) versus 75% (806/1071) were within the QC target ± 1 mm, for the first and second studies, respectively. One manufacturer (HiMedia) had major quality problems with 33% (26/78) of readings out of range in the first study and 17% (20/120) in the second study. Discs from some manufacturers showed unexpected variation in inhibition zone diameters (4-9 mm) for discs within the same vial. CONCLUSIONS: Antimicrobial discs from three of nine manufacturers exhibited excellent and reproducible quality. The discs of the other six manufacturers demonstrated various quality issues, some of which were severe. After presenting the results to manufacturers and users, all managed to improve the quality. Our study points to the need for more stringent criteria for disc manufacturing. Criteria should not only address the nominal potency of discs but also define the end result.

Development of EUCAST zone diameter breakpoints and quality control criteria for ceftazidime-avibactam 10-4 µg.

Ceftazidime-avibactam disk studies were performed for disk mass selection and for establishing EUCAST quality control ranges and zone diameter breakpoints. The disk mass study included disk diffusion testing with ceftazidime-avibactam 10-4 and 10-6 µg disks and broth microdilution MIC testing for challenge set of 94 Enterobacteriaceae and 45 Pseudomonas aeruginosa. EUCAST SOP 9.0-based QC and MIC-disk correlations studies were followed for development of ceftazidime-avibactam 10-4 µg ranges for Escherichia coli ATCC 25922, P. aeruginosa ATCC 27583, and Klebsiella pneumoniae ATCC 700603 and for zone diameter breakpoint determination. The ceftazidime-avibactam 10-4 and 10-6 µg disks performed similar in comparison to broth microdilution, with zones ≤ 14 mm for all resistant strains. The 10-4 µg disk was selected and used in QC and breakpoint studies. There was minimal variation of ceftazidime-avibactam 10-4 µg QC study results between disks, media, and sites. The QC ranges were within 7 mm for all strains. The zone diameter breakpoint study demonstrated good correlation of MIC and disk results. The established zone diameter breakpoints resulted in false susceptible rates of 1.6 and 4.0% for Enterobacteriaceae and P. aeruginosa. EUCAST selected the ceftazidime-avibactam 10-4 µg disk and established QC ranges for E. coli 25922 of 24-30 mm, P. aeruginosa ATCC 27853 of 21-27 mm, and K. pneumoniae ATCC 700603 of 18-24 mm. The zone diameter breakpoints that correlated best with the MIC breakpoints of susceptible ≤ 8 mg/L and resistant > 8 mg/L were Enterobacteriaceae (S ≥ 13, R < 13 mm) and P. aeruginosa (S ≥ 17, R < 17 mm).

Antimicrobial susceptibility testing of Kingella kingae with broth microdilution and disk diffusion using EUCAST recommended media.

OBJECTIVES: Increasing use of improved culture techniques and sensitive nucleic acid amplification assays have resulted in recognition of Kingella kingae as an important cause of invasive infections in young children, especially in septic arthritis, osteomyelitis, bacteraemia, and endocarditis. In 2016, EUCAST established clinical MIC breakpoints for K. kingae (published in EUCAST Clinical Breakpoint Tables v 7.0, 2017). The present study was carried out to produce MIC-zone diameter correlations for K. kingae on an international collection of isolates, with the aim of suggesting zone diameter breakpoints corresponding to the clinical MIC breakpoints. METHODS: Antimicrobial susceptibility testing was performed for 18 clinically relevant agents on a collection of 159 clinical isolates of K. kingae. Broth microdilution MIC determination and disk diffusion were performed according to EUCAST recommendations for fastidious organisms. RESULTS: The correlation between MICs and zone diameters was good for all agents with EUCAST breakpoints for K. kingae. ß-lactamase was detected in 41 isolates (26%) and these isolates were resistant to aminopenicillins. These isolates were also resistant to trimethoprim-sulfamethoxazole. Resistance to tetracyclines was detected in 8% of all isolates. All resistant isolates were correctly categorized for these agents with the proposed zone diameter breakpoints. One isolate, resistant to erythromycin but susceptible to other macrolides, was categorized as susceptible with erythromycin disk diffusion. No resistance was detected for the cephalosporins, carbapenems, and fluoroquinolones tested. CONCLUSION: Based on the results in this study, zone diameter breakpoints for K. kingae calibrated to EUCAST clinical MIC breakpoints were proposed and approved by EUCAST.

Antimicrobial susceptibility testing of colistin - evaluation of seven commercial MIC products against standard broth microdilution for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter spp.

OBJECTIVE: Both EUCAST and CLSI recommend broth microdilution (BMD) for antimicrobial susceptibility testing of colistin, but BMD is rarely used in routine microbiology laboratories. The objective of this study was to evaluate five commercially available BMD products and two brands of gradient tests for colistin MIC determination using BMD according to ISO standard 20776-1 as reference. METHODS: Colistin MIC determination was performed according to the manufacturer's instructions on five commercially available BMD products (Sensititre, MICRONAUT-S, MICRONAUT MIC-Strip, SensiTest, and UMIC) and two gradient tests (Etest and MIC Test Strip). Colistin reference MICs were determined using frozen panels according to ISO standard 20776-1. An international collection of Gram-negative bacteria (n=75) with varying levels of colistin susceptibility was tested. RESULTS: The colistin BMD products correlated well with reference tests, in particular for Sensititre and the two MICRONAUT products (essential agreement ≥96%: 66/69 (96%, CI 88-99%), 72/75 (96%, CI 88-99%) and 74/75 (99%, CI 92-100%)). The results were somewhat poorer for the BMD products SensiTest and UMIC: EA 88% (51/58, CI 77-95%) and 82% (61/74, CI 72-89%), respectively), and considerably poorer for the gradient tests (EA 43-71% depending on gradient test and Mueller-Hinton agar manufacturer). The gradient tests generally underestimated colistin MICs, resulting in a significant number of false susceptible results (9-18 of total 75 tests, compared with 1-3 for the BMD products). CONCLUSIONS: Based on the results of this study, we advise laboratories not to trust gradient tests for colistin susceptibility testing and to use broth microdilution methods for this purpose. There are several commercial broth microdilution tests available and in principle they perform well.

Emergence of clonally related multidrug resistant Haemophilus influenzae with penicillin-binding protein 3-mediated resistance to extended-spectrum cephalosporins, Norway, 2006 to 2013.

Resistance to cephalosporins in Haemophilus influenzae is usually caused by characteristic alterations in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene. Resistance to extended-spectrum cephalosporins is associated with high-level PBP3-mediated resistance (high-rPBP3), defined by the second stage S385T substitution in addition to a first stage substitution (R517H or N526K). The third stage L389F substitution is present in some high-rPBP3 strains. High-rPBP3 H. influenzae are considered rare outside Japan and Korea. In this study, 30 high-rPBP3 isolates from Norway, collected between 2006 and 2013, were examined by serotyping, multilocus sequence typing (MLST), ftsI sequencing, detection of beta-lactamase genes and minimum inhibitory concentration (MIC) determination. MICs were interpreted according to clinical breakpoints from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Respiratory isolates predominated (proportion: 24/30). The 30 isolates included one serotype f isolate, while the remaining 29 lacked polysaccharide capsule genes. Resistance to extended-spectrum cephalosporins (cefixime, 29 isolates/30 isolates; cefepime, 28/30; cefotaxime, 26 /30; ceftaroline, 26/30; ceftriaxone, 14/30), beta-lactamase production (11/30) and co-resistance to non-beta-lactams (trimethoprim-sulfamethoxazole, 13/30; tetracycline, 4/30; chloramphenicol, 4/30; ciprofloxacin, 3/30) was frequent. The N526K substitution in PBP3 was present in 23 of 30 isolates; these included a blood isolate which represents the first invasive S385T + N526K isolate reported from Europe. The L389F substitution, present in 16 of 30 isolates, coincided with higher beta-lactam MICs. Non-susceptibility to meropenem was frequent in S385T + L389F + N526K isolates (8/12). All 11 beta-lactamase positive isolates were TEM-1. Five clonal groups of two to 10 isolates with identical MLST-ftsI allelic profiles were observed, including the first reported high-rPBP3 clone with TEM-1 beta-lactamase and co-resistance to ciprofloxacin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole. Prior to this study, no multidrug resistant high-rPBP3 H. influenzae had been reported in Norway. Intensified surveillance of antimicrobial resistance is needed to guide empiric therapy.

Development of EUCAST zone diameter breakpoints and quality control range for Staphylococcus aureus with ceftaroline 5-µg disk.

This ceftaroline MIC/disk comparison study for Staphylococcus aureus was performed for the purpose of establishing EUCAST zone diameter breakpoints. Ceftaroline susceptibility for a challenge set of 70 methicillin resistant- and 30 methicillin susceptible-S. aureus was determined by 5-µg disk diffusion and broth microdilution methods. Seventeen isolates were retested by disk and MIC, and the remaining 83 isolates were retested by MIC. Molecular testing was performed on 19 isolates with borderline susceptible ceftaroline MIC results to assess any differences in mecA and epidemiological correlation. An additional set of 101 consecutive clinical S. aureus isolates were tested using the 5-µg disk. S. aureus ATCC 29213 was tested by multiple sites and media for QC range determination. Replicate MIC results were within ±1 doubling dilution, with tendency for slightly lower repeat MICs, and there was minimal variation in replicate zone results. Based on susceptible breakpoints for MIC of ≤1 mcg/mL and for disk of >20 mm, there was 100 % categorical agreement for 30 MSSA and 92 % categorical agreement for 70 MRSA. There were no common MLST or PBP changes for strains with MICs of 1 and 2 mcg/mL. All ceftaroline disk results for the consecutively collected isolates were >20 mm. EUCAST selected the ceftaroline 5-µg disk breakpoint of Susceptible ≥20, Resistant <20 mm because it correlated best with the MIC breakpoint of Susceptible ≤1, Resistant >1 mg/L. A ceftaroline 5-µg disk QC range for S. aureus ATCC 29213 of 24-30 mm was also established by EUCAST.

Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories.

With the support of ESCMID and European countries, EUCAST has developed a disk diffusion test with zone diameter breakpoints correlated with the EUCAST clinical MIC breakpoints. The development of the EUCAST disk diffusion method and quality control criteria are described, together with guidance on quality control and implementation of the method in clinical microbiology laboratories. The method includes the use of Mueller-Hinton agar without supplements for non-fastidious organisms and with 5% mechanically defibrinated horse blood and 20 mg/L ß-NAD for fastidious organisms, a standardized inoculum resulting in confluent growth, an incubation time of 16-20 h, a reading guide on how to read zone diameters on individual species-agent combinations and zone diameter breakpoints calibrated to the EUCAST clinical MIC breakpoints. EUCAST recommendations are described in detail and updated regularly on the EUCAST website (http://www.eucast.org).

Evaluation of the tissue toxicity of antiseptics by the hen's egg test on the chorioallantoic membrane (HETCAM).

BACKGROUND: Antiseptics are frequently used for the prophylaxis and treatment of local infections of chronic wounds. Whereas local antiseptics in general have a positive effect on wound healing an uncritical use may impair wound healing due to toxic side effects. OBJECTIVE: We sought to assess the vascular irritation potential of different antiseptic solutions and ointments commonly used for short and long term application as a measure of tissue toxicity. METHOD: The vascular irritation was evaluated by the hen's egg test (HET) on the chorioallantoic membrane (CAM). The effects on the vessels of a mucous membrane were directly assessed by stereomicroscopic observation in vivo. RESULTS: Severe CAM irritation was observed after short-term applications of 1% octenidin-2HCl (Octenisept), 72% isopropanol (Cutasept), 0.35% chloroxylenol (Dettol) and 10% PVP-I ointment (Betaisodona). Medium irritations were observed for 10% PVP-I solution (Betaisodona), 3% lysosomal PVP-I ointment (Repithel), 1.8% cadexomer-iodine ointment (Iodosorb) and 1% cadexomer-iodine pellets (Iodosorb). Finally, slight irritations were observed for 1% PVP-I solution (Betaisodona), 0.1% polyhexanid plus betain (Prontosan) and 1% silver-sulfadiazine ointment (Flammazine), whereas 0.04% polyhexanid solution (Lavanid), washings from sterile maggots of Lucilia sericata and filtrated enzymes from Clostridium histolyticum (Iruxol-N) showed no effects of irritation. In the long-term approaches, no vascular irritations were found for polyhexanid, washings from Lucilia sericata and enzyme filtrations from Clostridium histolyticum. CONCLUSION: The vascular injuries caused by the studied antiseptics are an indirect indicator of their tissue toxicity. Strikingly, even therapeutic substances, which have been regarded as safe in their application for the treatment of chronic wounds in clinical studies, showed severe irritations on the CAM. We suggest that agents with no or low irritation potential on the CAM should be preferred in the clinical practice in order to obtain optimal results.

Oxidation of polyphenols in phytate-reduced high-tannin cereals: effect on different phenolic groups and on in vitro accessible iron.

After reduction of phytate with phytase, water slurries of two high-tannin cereal flours were incubated with polyphenol oxidase (mushroom tyrosinase), and the effects on different phenolic groups and on in vitro accessible iron were studied. Enzyme incubation was also performed after cooking, soaking, and germination of the cereals. Phytase incubation significantly decreased the phytate content, and incubation with polyphenol oxidase had a reducing effect on the total phenol content, as well as on the amount of catechol and resorcinol groups. The in vitro accessible iron increased when the cereals were incubated with phytase and polyphenol oxidase, and the highest accessibility of iron was obtained when the germinated samples were incubated. The results from this study imply that oxidation of polyphenols in high-tannin cereals, after reduction of phytate, may be used to increase the bioavailability of iron in foods prepared from these cereals.