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Objectives: To evaluate the performance of an in-house developed disk diffusion method for aztreonam in combination with avibactam against Enterobacteriales., Methods: The in vitro antibacterial activity of aztreonam with avibactam against 204 carbapenemase-producing Enterobacteriales was determined by a disk diffusion method, with a broth microdilution method as a reference., Results: The optimal S/R breakpoints for disk diffusion tests of 30/20 and 10/4 µg disks, calculated by the dBETs software using the model-based approaches, were ≥22/≤21 and ≥12/≤11 mm, respectively. On the basis of the estimated breakpoints, the CAs for disk diffusion tests of 30/20 and 10/4 µg aztreonam/avibactam disks were both 98.0%, with 0.5% major error and 37.5% very major error., Conclusions: The home-made disk diffusion method is an economical and practical method for clinical microbiology laboratories to determine the antibacterial susceptibility of aztreonam with avibactam against Enterobacteriales., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The objective of this research was to evaluate the correlation between inhibitory zones and MIC when testing ceftazidime-avibactam using disk diffusion, Etest, and broth microdilution method established by the Clinical and Laboratory Standards Institute (CLSI). Four-hundred and 58 isolates of Enterobacterales isolated from 54 medical centers from the China Antimicrobial Surveillance Network (CHINET) in 2016 to 2020 were collected. Antimicrobial susceptibility testing using broth microdilution, Etest, and disk diffusion were performed according to the CLSI. Of the 458 Enterobacterales , 17.2% (79/458) and 82.8%(379/458) were resistant or susceptible to ceftazidime-avibactam by broth microdilution, respectively. Compared with the broth microdilution method, the categorical agreement (CA) and essential agreement (EA) of the Etest were 99.6% (456/458) and 94.8% (434/458), respectively; the major error (ME) and very major error (VME) were both 0.2% (1/458). For disk diffusion, the CA and VME were 99.8% (457/458) and 0.2% (1/458), respectively. For Escherichia coli, the CA and EA of the Etest were 100% and 97.1% (135/139), respectively. The CA of the disk diffusion was 100%. For Klebsiella pneumoniae, the CA and EA of the Etest were 99.3% (288/290) and 93.4% (271/290), respectively, the ME and VME were both 0.3% (1/290). The CA and VME of disk diffusion were 99.7% (289/290) and 0.3% (1/290), respectively. For other Enterobacterales , the CA and EA of the Etest were 100% and 96.6% (28/29), respectively. The CA of the disk diffusion was 100%. Ceftazidime-avibactam disk diffusion (30/20-μg disks) and Etest demonstrated good performance for ceftazidime-avibactam susceptibility testing against Enterobacterales clinical isolates. IMPORTANCE Multidrug-resistant Gram-negative bacteria, especially for extended-spectrum β-lactamases-producing and carbapenemase-producing Enterobacterales , are disseminating rapidly around the world. Treatment options for these infections are limited, which prompt the development of novel or combinational therapies to combat the infections caused by multidrug-resistant pathogens. The newly available β-lactam combination agent ceftazidime-avibactam has been demonstrated good in vitro and in vivo activity against ESBL, AmpC, KPC-2, or OXA-48-like-producing isolates and has shown promise in treating carbapenem-resistant Enterobacterales infections. Concerningly, there are few available automated systems for ceftazidime-avibactam susceptibility testing, and the broth microdilution method is hard to perform in most routine laboratories. Therefore, we urgently need an economical and practical method for the accurate detection of ceftazidime-avibactam activity against Gram-negative bacilli. Here, we evaluate the performance of the disk diffusion and Etest compared with the reference broth microdilution method against Enterobacterales clinical strains.

Plazomicin (PLZ), brand name ZEMDRI (Cipla Therapeutics), is a novel aminoglycoside antibiotic approved by the U.S. Food and Drug Administration (FDA) for treatment of complicated urinary tract infections including pyelonephritis. ETEST® is a gradient diffusion method that represents an alternative to the more laborious broth micro-dilution (BMD) method for performing antimicrobial susceptibility testing (AST). A multi-center evaluation of the performance of the new ETEST PLZ (bioMérieux) was conducted in comparison with BMD following FDA and International Standards Organization (ISO) recommendations using FDA-defined breakpoints. Clinical isolates of Enterobacterales ( n  = 598) were included. Fifty-three isolates were resistant to PLZ according to BMD. Overall, the ETEST PLZ demonstrated 99.0% essential agreement (EA), 92.8% category agreement (CA), 1.9% very major errors (VME), 0% major errors (ME), and 7.0% minor errors (mE) with both clinical and challenge isolates of Enterobacterales. The VME was found for a single Serratia marcescens strain. Individual species demonstrated EA rates ≥ 90%. In conclusion, we report that ETEST PLZ represents an accurate tool for performing PLZ AST of Enterobacterales.

Background: Over the past decade, drug resistance pattern has worsened for many of the uropathogens due to overuse of antibiotics for empiric treatment. The burden of extended spectrum beta-lactamase (ESBL) producing Enterobacteriaceae associated urinary tract infections (UTI) has become increasingly more common, limiting treatment options among children presenting with febrile UTI. We investigated the burden and correlates of ESBL producing Enterobacteriaceae associated UTI among children and antibacterial resistance pattern., Methods: 284 midstream urine specimens were collected using standard aseptic techniques from 284 children who were diagnosed with suspected UTI. Urine culture and bacteria isolation were performed following standard bacteriological techniques. The Kirby-Bauer disk diffusion technique and the double-disc synergy test were used to investigate antibiotic susceptibility and presence of ESBL production., Results: UTI was confirmed using a positive urine culture for a relevant pathogen in 96/284 (33.8%) of the cases. Enterobacteriaceae accounted for 75% (72/96) of etiologies of UTI in children. The most frequent Enterobacteriaceae spp. were E. coli , 44.4% (32/72) and K. pneumonia , 27.8% (20/72). The overall multidrug resistance rate was 86.1% (62/72). ESBL-producers accounted for 41.7% (30/72) of the isolated Enterobacteriaceae . ESBL producing K. pneumonia and E. coli isolates accounted for 70% (14/20) and 37.5% (12/32), respectively. History of UTI in the past 1 year (adjusted odds ratio (AoR) = 0.08, 95%CI (0.01 - 0.57)) and medium family wealth index (AoR = 0.03, 95%CI (0.00 - 0.27)) protected from infection with ESBL-producing Enterobacteriaceae. Conclusion . ESBL production was more common in K. pneumonia and appeared to be a major factor contributing drug resistance UTI in children. The findings call for the need to incorporate ESBL testing in the routine clinical practice. The resistance level to commonly prescribed first-line antibiotics observed within Enterobacteriaceae was alarming calling for strengthened antimicrobial stewardship., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Asnakech Agegnehu et al.)

Determination of polymyxins susceptibility by clinical laboratories is a nightmare, mainly because of physicochemical properties of the drug. Elution tests have already been proposed for colistin, but not for polymyxin B. We aimed to evaluate accuracy of Polymyxin B broth disk elution (PBDE) to determine the susceptibility to this drug. We evaluated 196 Enterobacterales (45.9% polymyxin B-resistant). PBDE was done in 15-mL cation-adjusted Mueller-Hinton broth where one polymyxin B disk (300 U) was eluted (2 μg/mL). BMD was performed as reference method. Categorical Agreement (CA), Major Error (ME) and Very Major Error (VME) were 99.5%, 0% and 1.11% (one false-negative K. pneumoniae MIC 4 μg/mL), respectively. As some institutions preferably use polymyxin B over colistin and in some countries colistin are not commercially available, to specifically evaluate polymyxin B is important. PBDE proved to be a cheap and easy to perform methodology to evaluate susceptibility to polymyxin B among Enterobacterales., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Ceftazidime-avibactam was approved in China in 2019 for treating complicated intra-abdominal infections, hospital-acquired pneumonia, ventilator-associated pneumonia, and infections caused by Enterobacterales and Pseudomonas aeruginosa for which treatment options are limited. However, no currently available commercial systems have been approved for antimicrobial susceptibility testing of ceftazidime-avibactam in China. Here, we evaluated the Etest and disk diffusion method for detecting the activity of ceftazidime-avibactam against Enterobacterales and P. aeruginosa in China., Results: In total, 194 Enterobacterales and 77 P. aeruginosa isolates, which were divided into a random selection group (140 Enterobacterales and 46 P. aeruginosa isolates) and stock group (54 Enterobacterales and 31 P. aeruginosa isolates), were assessed by the Etest, disk diffusion and broth microdilution methods. Minimum inhibitory concentrations and zone diameters were interpreted according to the CLSI supplement M100 30th edition. For all 271 tested isolates, no very major errors were found by using Etest, whereas the overall major error rate was 2.0% (4/203). The overall categorical agreement rates of Etest for Enterobacterales and P. aeruginosa were 99.5% (193/194) and 96.1% (74/77), respectively, and the essential agreement rates were 95.9% (186/194) and 94.8% (73/77), respectively. The disk diffusion method showed that the very major error and major error rates were 1.5% (3/204) and 2.5% (5/203), respectively. Overall categorical agreement rates values of the disk diffusion method for Enterobacterales and P. aeruginosa were 98.5% (191/194) and 93.5% (72/77) compared with broth microdilution, respectively., Conclusions: For Enterobacterales and P. aeruginosa, both the Etest and disk diffusion method showed acceptable performance as alternatives to the standard broth microdilution method for clinical treatment interpretation. Application of the disk diffusion method in Enterobacterales was slightly better than that in P. aeruginosa.

Piperacillin-tazobactam (P/T) is a β-lactam-β-lactamase inhibitor combination frequently used in the hospital setting. Etest is a gradient diffusion method that represents an alternative to broth microdilution (BMD) for performing antimicrobial susceptibility testing. We conducted a multicenter evaluation of the performance of the new P/T Etest compared to that of BMD following U.S. Food and Drug Administration (FDA) and International Standards Organization (ISO) standard ISO 20776-2 criteria using Clinical and Laboratory Standards Institute (CLSI)-FDA and European Committee on Antimicrobial Susceptibility Testing (EUCAST) interpretive breakpoints, respectively. A total of 977 isolates (775 Enterobacterales isolates, 119 Pseudomonas aeruginosa isolates, and 83 Acinetobacter baumannii complex isolates) were tested. Overall essential agreement (EA) was 96.4% and 96.6% for Enterobacterales when FDA and ISO 20776-2 criteria, respectively, were followed. EA was 98.3% for P. aeruginosa and 91.6% for the A. baumannii complex when both the FDA and ISO criteria were followed. Applying CLSI-FDA breakpoints, categorical agreement (CA) reached 93.0%, 93.3%, and 89.2% for the Enterobacterales , P. aeruginosa , and the A. baumannii complex, respectively. Two very major errors (VMEs; 1.1%) were found among the Enterobacterales (for 2 Klebsiella pneumoniae isolates). No additional major errors (MEs) or VMEs were found. Applying EUCAST breakpoints, CA was 94.8% and 95.8% for Enterobacterales and P. aeruginosa , respectively (no breakpoints are currently available for the A. baumannii complex). No VMEs were observed among the Enterobacterales , but 2 (0.4%) MEs were found. Among the P. aeruginosa isolates, 2 (6.9%) VMEs and 3 (3.3%) MEs were observed. These errors resulted when P/T Etest MICs were 1 doubling dilution apart from the BMD MICs. In conclusion, the new P/T Etest represents an accurate tool for performing antimicrobial susceptibility testing of Enterobacterales , P. aeruginosa , and A. baumannii complex isolates with limited category errors., (Copyright © 2020 American Society for Microbiology.)

Colistin and polymyxin B are old drugs that have been reintroduced to treat Gram-negative infections lacking other treatment options; however, colistin resistance have been reported. To know the correct susceptibility pattern is mandatory for multidrug resistant bacteria. Broth microdilution method is the gold standard to evaluate colistin and polymyxin B susceptibility; nevertheless, it is time consuming and needs expertise to be performed. Disk diffusion method on Müeller-Hinton agar is no longer recommended to evaluate polymyxins susceptibility. In this study we evaluated two methods (disk diffusion in broth and broth macrodilution) as alternative options to identify polymyxin resistance in an easy way. A total of 536 Enterobacteriales isolates were assessed for colistin susceptibility. All non-wild type Enterobacteriales (41) were chosen and 31 wild type bacteria were randomly selected, were used to perform disk diffusion tests in broth and for broth macrodilution tests. We found 100% of concordance between both tested methods and broth microdilution. In conclusion, these two methods are reliable and easier options that complement as initial screening susceptibility for colistin in Enterobacteriales in microbiology laboratories lacking personnel and infrastructure to perform broth microdilution method., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A phenotypic assay based on colistin pre-diffusion and differential inhibition of Mobile Colistin Resistance (MCR) protein activity by EDTA showed that, of the 92 strains tested, all MCR producers (49) exhibited an increase ≥5 mm in the inhibition zone around the area of pre-diffusion in the presence of EDTA, in comparison with colistin alone. Results suggest that CPD-E may differentiate MCR-producing microorganisms from resistant microorganisms without this marker., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Third-generation cephalosporin resistance among Enterobacteriaceae , mediated by the spread of extended-spectrum β-lactamases (ESBLs), is a very serious medical concern with limited therapeutic options. Enmetazobactam (formerly AAI101) is a novel penicillanic sulfone β-lactamase inhibitor active against a wide range of ESBLs. The combination of enmetazobactam and cefepime has entered phase 3 development in patients with complicated urinary tract infections. Using the Clinical and Laboratory Standards Institute (CLSI) M23 tier 2 study design, broth microdilution MIC and disk diffusion quality control (QC) ranges were determined for cefepime-enmetazobactam. Enmetazobactam was tested at a fixed concentration of 8 μg/ml in the MIC assay, and a cefepime-enmetazobactam disk mass of 30/20 μg was used in the disk diffusion assay. Escherichia coli ATCC 25922, E. coli ATCC 35218, E. coli NCTC 13353, Klebsiella pneumoniae ATCC 700603, and Pseudomonas aeruginosa ATCC 27853 were chosen as reference strains. The CTX-M-15-producing E. coli NCTC 13353 isolate is recommended for routine testing to control for inhibition of ESBL activity by enmetazobactam. Broth microdilution MIC QC ranges spanned 3 to 4 doubling dilutions and contained 99.6% to 100.0% of obtained MIC values for the five reference strains. Disk diffusion yielded inhibition zone diameter QC ranges that spanned 7 mm and encompassed 97.1% to 100.0% of the obtained values. Quality control ranges were approved by the CLSI in 2017 (broth microdilution MIC) and 2019 (disk diffusion). The established QC ranges will ensure that appropriate assay performance criteria are attained using CLSI reference methodology when determining the susceptibility of clinical isolates to cefepime-enmetazobactam., (Copyright © 2019 American Society for Microbiology.)

We evaluated several EDTA-based combined-disk tests to detect 25 mcr producers among 48 Enterobacteriaceae. Colistin disks plus EDTA (292/584 μg) on MH and CAMH agar were used. Results were positive if with chelator there was an inhibition zone increase ≥3 mm compared to colistin alone. All tests resulted unreliable (sensitivity ≤68%)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ceftolozane-tazobactam (C/T) is a novel beta-lactam-beta-lactamase inhibitor combination antibiotic approved by the U.S. Food and Drug Administration in 2014 for the treatment of complicated intra-abdominal infections (in combination with metronidazole) and complicated urinary tract infections. In this study, we evaluated the performance of the C/T Etest, a gradient diffusion method. C/T Etest was compared to broth microdilution (BMD) for 51 Enterobacteriaceae challenge isolates and 39 Pseudomonas aeruginosa challenge isolates at three clinical sites. Essential agreement (EA) between the methods ranged from 47 to 49/51 (92.2 to 96.1%) for the Enterobacteriaceae , and categorical agreement (CA) ranged from 49 to 51/51 (96.1 to 100.0%). EA and CA for P. aeruginosa were 100% at all sites. The C/T Etest was also compared to BMD for susceptibility testing on 966 clinical isolates (793 Enterobacteriaceae , including 167 Klebsiella pneumoniae and 159 Escherichia coli isolates, in addition to 173 P. aeruginosa isolates) collected at four clinical sites. EA between Etest and BMD was 96.9% for Enterobacteriaceae isolates and 98.8% for P. aeruginosa isolates. Within the Enterobacteriaceae , isolates from each species examined had >96% CA. For the clinical isolates, no very major errors were identified but two major errors were found (one for K. pneumoniae and one for Providencia rettgeri ). By BMD, 47.0% of Enterobacteriaceae and 46.2% of P. aeruginosa challenge strains were nonsusceptible to C/T by CLSI breakpoint criteria; 8.2% of clinical Enterobacteriaceae isolates and 12.1% of clinical P. aeruginosa isolates were nonsusceptible to C/T by CLSI breakpoint criteria. In conclusion, Etest is accurate and reproducible for C/T susceptibility testing of Enterobacteriaceae and P. aeruginosa ., (Copyright © 2018 American Society for Microbiology.)

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).

We evaluated the correlation between MIC and disk diffusion inhibition zones when testing ceftazidime-avibactam, using the 30/20-μg disk and the disk diffusion and MIC breakpoints established by the U.S. FDA and the Clinical and Laboratory Standards Institute (CLSI). Organisms used included 2 groups of Enterobacteriaceae isolates and 2 groups of Pseudomonas aeruginosa isolates; 1 group of each consisted of randomly selected isolates and the second group consisted of a challenge group from thousands of surveillance isolates with an increased proportion of organisms displaying ceftazidime-avibactam MIC values close to the breakpoints. Broth microdilution, disk diffusion tests, and data analysis were performed according to reference standardized methods. Ceftazidime-avibactam breakpoints of ≤8/4 (susceptible) and ≥16/4 μg/ml (resistant) for MIC and ≥21/≤20 mm for disk diffusion, as established by the U.S. FDA and the CLSI, were applied for Enterobacteriaceae and P. aeruginosa Ceftazidime-avibactam MIC and disk zone (30/20-μg disk) correlation were acceptable when testing Enterobacteriaceae (overall, very major [VM] and major [Ma] error rates of 0.4% and 0.0%, respectively) and nearly so when testing P. aeruginosa (2.3% VM and 2.9% Ma errors). In summary, disk diffusion and broth microdilution testing results demonstrated good categorical agreement for ceftazidime-avibactam against Enterobacteriaceae and P. aeruginosa , using 30/20-μg disks., (Copyright © 2018 American Society for Microbiology.)

The combination disk diffusion extended-spectrum β-lactamase (ESBL) confirmation test (CDT) is used for the confirmation of ESBL production in Enterobacteriaceae and usually takes 16-20 h to results. In this study, we searched for the shortest possible incubation time without a reduction in reliability. A total of 125 ESBL screening-positive isolates were subjected to CDT and were molecularly characterised by microarray. Inhibition zones were read every hour over 6-18 h of incubation. Concordance between earlier and 18-h readings was calculated for each hour. Results were validated on 224 isolates during routine clinical practice. For the initial 125 isolates, concordance (Cohen's κ) between the 6-h and 18-h readings was 0.88 [95% confidence interval (CI) 0.78-0.96; P <0.001]. The earliest time point for full concordance with the 18-h reading was 10 h. Validation of the 10-h reading for 224 clinical isolates resulted in a concordance of 0.99 (95% CI 0.98-1.0) between the 10-h and 18-h readings. Overall concordance on all 349 isolates was 0.99 (95% CI 0.97-1.0). Reading after 10 h of incubation has an excellent correlation with results after 18 h of incubation. This can significantly reduce the turnaround time for ESBL detection in laboratories with long opening hours or providing a 24/7 service. Consequently, there is a potential for implementing infection control measures up to 8 h earlier., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

A promising means of rapid screening of extended-spectrum-β-lactamase (ESBL), AmpC β-lactamase, and co-production of ESBL and AmpC that combines resazurin chromogenic agar (RCA) with a combined disc method is here reported. Cefpodoxime (CPD) discs with and without clavulanic acid (CA), cloxacillin (CX) and CA+CX were evaluated against 86 molecularly confirmed β-lactamase-producing Enterobacteriaceae, including 15 ESBLs, 32 AmpCs, nine co-producers of ESBL and AmpC and 30 carbapenemase producers. The CA and CX synergy test successfully detected all ESBL producers (100% sensitivity and 98.6% specificity) and all AmpC producers (100% sensitivity and 96.36% specificity). This assay also performed well in screening for co-existence of ESBL and AmpC (88.89% sensitivity and 100% specificity). The RCA assay is simple and inexpensive and provides results within 7 hr. It can be performed in any microbiological laboratory, in particular, in geographic regions in which ESBL, AmpC or co-β-lactamase-producing Enterobacteriaceae are endemic., (© 2017 The Societies and John Wiley & Sons Australia, Ltd.)

Object: This surveillance aimed to investigate the antimicrobial resistance profiles of Enterobacteriaceae pathogens in Southern China during 2001-2015., Methods: A total of 6858 Enterobacteriaceae isolates were collected, including 4276 E. coli, 1992 K. pneumoniae and 590 Enterobacter spp. Disk diffusion method and minimum inhibitory concentrations method were used for susceptibility testing, with results interpreted by the CLSI (2015)., Results: Urinary tract remained the dominant isolated site among E. coli (49.88%), whereas 53.26% K. pneumoniae and 45.25% Enterobacter spp. were from Sputum. The carbapenems maintained the highest antimicrobial activity (resistance rates <15%), followed by piperacillin-tazobactam and amikacin. Gentle increases were obtained in carbapenems-resistant K. pneumoniae and Enterobacter spp. (eg. from 4.5% to 11.2% and 3.2% to 14.5% in imipenem, repestively). The third-generation cephalosporins showed high and stable resistance among Enterobacteriaceae pathogens during the studied period, with ceftazidime as the most active third-generation cephalosporin against Enterobacteriaceae. Isolates from ICU department showed higher or similar resistance rates among Enterobacteriaceae pathogens compared to other wards., Conclusion: Carbapenems are the most potent antibiotic agents against Enterobacteriaceae pathogens. Due to the complicated susceptibility profiles, prescribing guidelines should be based on the knowledge of antibiogram of pathogens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The standard incubation time for antimicrobial susceptibility testing (AST) by disk diffusion is primarily based on laboratory working hours rather than growth and kill characteristics of bacteria. Faster AST results could result in better patient outcomes and reduced costs by initiating earlier appropriate therapy. The earliest possible reading moment for disk diffusion using established disk zone diameter breakpoints for Enterobacteriaceae was determined with a special focus on the accuracy of the results. A total of 88 Enterobacteriaceae challenge isolates, including isolates with specific resistance mechanisms such as extended-spectrum β-lactamase (ESBL), were subjected to disk diffusion with 15 antibiotics. Hourly images were automatically produced by an incubator/camera combination from 1 h to 20 h. Disk zones were plotted over time for all combinations. Essential and categorical agreement rates using the final reading after 20 h of incubation as a reference were calculated for every hour. In total, 1320 antibiotic-micro-organism combinations were tested. Clear growth with readable inhibition zones was visible after 6 h of incubation for the majority (95.8%) of plates and after 7 h for all incubated plates. However, zone sizes changed significantly after those time points for a number of strains. After 10 h of incubation, minor, major and very major error rates were 1.6% (n = 21), 0.2% (n = 1) and 0.7% (n = 4), respectively. The results of this study clearly indicate that early reading of inhibition zones to 10 h after incubation is feasible and accurate and thus may save significantly on turnaround time., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Objectives: The aim of this study was to validate the use of LML antimicrobial gradient strips for quantitative determination of carbapenem susceptibility in Enterobacteriaceae., Methods: A total of 95 non-redundant Enterobacteriaceae strains isolated during 2012-2014 were used for this validation study. Initially, LML antimicrobial gradient strips were validated for their performance in comparison with the agar dilution method. The test strip was then validated in comparison with broth microdilution (BMD) and Etest with 24 selected strains using the same inocula and other laboratory parameters., Results: The LML strip showed 83%, 68% and 86% essential agreement (within ±1 log 2 dilution) with the reference methods of agar dilution, BMD and Etest, respectively; furthermore, essential agreement was >90% within ±2 log 2 dilution. Categorical agreement was ≥87% with all reference methods according to Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. However, the meropenem strip requires performance improvements to fulfil US Food and Drug Administration (FDA) and International Organization for Standardization (ISO) requirements for an antimicrobial susceptibility test device., Conclusions: In LML antimicrobial gradient strip minimum inhibitory concentrations (MICs) were comparable with Etest MICs and it might serve as a reasonable, cost-effective alternative to Etest for quantitative determination of carbapenem susceptibility in Enterobacteriaceae., (Copyright © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

OXA-48 is the most prevalent carbapenemase in Enterobacteriaceae in Europe and the Middle East, but it is frequently missed because many isolates display low MICs for carbapenems. Furthermore, in contrast to metallo-β-lactamases or Klebsiella pneumoniae carbapenemases (KPC), no specific inhibitor is available for the phenotypic detection of OXA-48. Molecular detection of bla OXA-48 is the "gold standard" but is not available in many laboratories. A few phenotypic assays have been described but have not been independently evaluated. The aim of this study was the systematic comparison of phenotypic tests and an immunochromatographic assay (ICT) for the detection of OXA-48/OXA-48-like carbapenemases and the development of an algorithm for reliable phenotypic detection of OXA-48. Four phenotypic tests (temocillin disk test, faropenem disk test, OXA-48 disk test, and high-inoculum [HI] OXA-48 disk test) and a new ICT (OXA-48 K -SeT) were compared by using a set of 166 Enterobacteriaceae isolates, including isolates producing OXA-48/OXA-48-like carbapenemases ( n = 84) or Ambler class A and B carbapenemases ( n = 41) and carbapenemase-negative isolates ( n = 41). The sensitivity and specificity for the different assays were 100% and 43.9% for temocillin, 57.1% and 98.8% for faropenem, 53.6% and 100% for the OXA-48 disk test, 98.8% and 97.6% for the HI OXA-48 disk test, and 100% and 100% for the ICT, respectively. The ICT displayed the highest sensitivity and specificity and was the most rapid assay, but it is more costly than phenotypic assays. Based on these results, a new algorithm incorporating temocillin, faropenem, and ICT which allows cost-effective detection of OXA-48 with 100% sensitivity and specificity was developed., (Copyright © 2017 American Society for Microbiology.)

In this study, we aimed to identify the molecular carbapenemase types of the Enterobacteriaceae isolates and to evaluate the performance of manually prepared and commercially available combination disc methods and the modified Hodge test. One hundred and forty carbapenemase producing isolates and 45 isolates as control group were included in our study. The Xpert CARBA-R test was used as the molecular method. Antibiotic susceptibility tests were performed using combined discs, manually prepared with APBA (3-aminophenyl boronic acid), DPA (dipicolinic acid), EDTA (Ethylene diamine tetra acetic acid), cloxacillin supplements and Mastdiscs Combi-D70C that includes four antibiotic discs with specific inhibitors and temocillin discs. The modified Hodge test was performed on all isolates. OXA-48 gene was identified in 129 isolates , the NDM gene was identified in 10 isolates and VIM in one isolate. Thirty inaccurate results (30/185, 16%) were detected by using the manually prepared confirmation test. The sensitivity and specificity of this test were identified respectively 85% and 73%. Also, the sensitivity and specificity of the Mastdiscs Combi-D70C were identified as 100%. Negative results were detected in 3 NDM isolates with the use of a modified Hodge test. Sensitivity and specificity were calculated for the modified Hodge test respectively 97% and 100%. Finally, molecular methods provide results rapidly but they are not always easily accessible. The modified Hodge test can be used only for screening as a first step test and is not one of the tests that can identify the type of the carbapenemase. When carbapenem-resistant Enterobacteriaceae are detected, a commercial kit like Mastdiscs Combi-D70 may be preferred instead of the manually prepared phenotypic verification tests., (Copyright © 2016 Elsevier B.V. All rights reserved.)

ChromID ESBL agar and Brilliance ESBL agar were compared for the isolation of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae from 298 stools. These had comparable sensitivity and selectivity for the 116 positive samples. Pre-enrichment with cefpodoxime and extending incubation to 48 hours after direct plating both significantly increased sensitivity but reduced selectivity of both agars., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: The objective of this study was to assess the performance of an algorithm based on the disc diffusion method for the screening of carbapenemase-producing Enterobacteriaceae (CPE) referred to the French National Reference Centre for Antibiotic Resistance., Methods: From April to June 2014, all isolates of Enterobacteriaceae referred to the French National Reference Centre for Antibiotic Resistance were included. The inhibition zone diameters of imipenem, ticarcillin/clavulanate and temocillin using EUCAST disc diffusion methodology were recorded. All isolates were subjected to the algorithm proposed by the Antibiogram Committee of the French Society of Microbiology (CA-SFM) for the screening of carbapenemase producers. Phenotypic, biochemical and molecular detection of carbapenemases was performed for all isolates., Results: A total of 621 consecutive enterobacterial isolates with decreased susceptibility to carbapenems were tested. They included 213 CPE [OXA-48-like (n = 183), NDM (n = 22), VIM (n = 3), KPC (n = 3) and OXA-48-like + NDM (n = 2)] and 408 non-carbapenemase producers. The CA-SFM algorithm combining cut-off values of 15 mm, 15 mm and 22 mm for ticarcillin/clavulanate, temocillin and imipenem, respectively, perfectly detected 204 isolates (32.8%) as non-carbapenemase producers, leading to a negative predictive value of 100% for this algorithm., Conclusions: Implementation of the CA-SFM algorithm in clinical microbiology laboratories may avoid additional testing for CPE in one-third of the enterobacterial isolates with decreased susceptibility to carbapenems., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The current phenotypic methods for detecting carbapenemase-producing Enterobacteriaceae (CPE) allow differentiation between class A and B carbapenemases, but they cannot confirm in a single test class D OXA-48 carbapenemase producers. In this study, we evaluated a new phenotypic test, the OXA-48 disk test, which is based on an imipenem disk and two blank disks adjacent to the imipenem disk, loaded with the tested strain and impregnated with EDTA and EDTA plus phenyl boronic acid (PBA), respectively. The evaluation of the OXA-48 disk test was performed with 81 genotypically confirmed OXA-48-type-producing Enterobacteriaceae isolates (41 extended-spectrum β-lactamase [ESBL] producers, 3 AmpC producers, and 37 non-ESBL, non-AmpC producers). To measure the specificity of the test, 173 genotypically confirmed OXA-48-negative Enterobacteriaceae isolates (57 Klebsiella pneumoniae carbapenemase [KPC] producers, 34 VIM producers, 23 KPC/VIM producers, 22 NDM producers, and 37 AmpC or ESBL producers and porin deficient) that were nonsusceptible to at least one carbapenem were chosen for testing. Using the imipenem disk and the distortion of the inhibition halo around both blank disks containing EDTA and EDTA/PBA, the test differentiated all but 3 of the 81 OXA-48 producers (sensitivity of 96.3%). The test was negative for OXA-48 production in all but 4 of the 173 carbapenem-nonsusceptible isolates producing other carbapenemases, AmpCs, or ESBLs (specificity of 97.7%). This evaluation shows that the OXA-48 disk test is an accurate phenotypic method for the direct differentiation of OXA-48-producing Enterobacteriaceae. Its use along with combined disk tests employing inhibitor-supplemented carbapenem disks might allow the differentiation of the currently known carbapenemase types in Enterobacteriaceae species and provide important infection control information., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: We evaluated the combined use of the modified Hodge test (MHT) and carbapenemase inhibition test (CIT) using phenylboronic acid (PBA) and EDTA to detect carbapenemase-producing Enterobacteriaceae (CPE) and metallo-β-lactamase (MBL)-producing Pseudomonas spp., Methods: A total of 49 isolates of CPE (15 Klebsiella pneumoniae carbapenemase [KPC], 5 Guiana extended-spectrum β-lactamase [GES]-5, 9 New Delhi metallo-β-lactamase [NDM]-1, 5 Verona integron-encoded metallo-β-lactamase [VIM]-2, 3 imipenem-hydrolyzing β-lactamase [IMP], and 12 oxacillinase [OXA]-48-like), 25 isolates of MBL-producing Pseudomonas spp. (14 VIM-2 and 11 IMP), and 35 carbapenemase-negative controls were included. The MHT was performed for all isolates as recommended by the Clinical and Laboratory Standards Institute. Enhanced growth of the indicator strain was measured in mm with a ruler. The CIT was performed by directly dripping PBA and EDTA solutions onto carbapenem disks that were placed on Mueller-Hinton agar plates seeded with the test strain., Results: Considering the results of the MHT with the ertapenem disk in Enterobacteriaceae and Pseudomonas spp., the CIT with the meropenem disk in Enterobacteriaceae, and the imipenem disk in Pseudomonas spp., three combined disk tests, namely MHT-positive plus PBA-positive, EDTA-positive, and MHT-positive plus PBA-negative plus EDTA-negative, had excellent sensitivity and specificity for the detection of KPC- (100% sensitivity and 100% specificity), MBL- (94% sensitivity and 100% specificity), and OXA-48-like-producing isolates (100% sensitivity and 100% specificity), respectively., Conclusions: Combined use of the MHT and CIT with PBA and EDTA, for the detection of CPE and MBL-producing Pseudomonas spp., is effective in detecting and characterizing carbapenemases in routine laboratories.

We performed extended-spectrum beta-lactamase (ESBL) phenotypic testing and molecular characterization of three ESBL genes (TEM, SHV and CTX-M) and susceptibility testing by Clinical Laboratory Standards Institute (CLSI) disk diffusion method against three cephalosporins (ceftriaxone, ceftazidime, cefepime) and a cephamycin (cefoxitin) among 128 Thai Escherichia coli and 84 Thai Klebsiella pneumoniae clinical isolates. ESBL production was discovered in 62% of E. coli and 43% of K. pneumoniae isolates. All isolates susceptible to ceftriaxone were ESBL-negative. Nearly all isolates non-susceptible to ceftriaxone, ceftazidime and cefepime produced ESBL; the presence of CTX-M genes in the isolates correlated with a ceftriaxone non-susceptible phenotype. Thirty-nine of 83 isolates (47%) of ceftazidime-susceptible E. coli and 50 of 99 isolates (50.5%) of cefepime-susceptible E. coli were ESBL-producing. SHV-type beta-lactamase genes were more prevalent among K. pneumoniae than E. coli isolates. CTX-M was the major ESBL gene harbored by ESBL-producers in both E. coli and K. pneumoniae isolates. Non-CTX-M ESBL-producers were found only among K. pneumoniae isolates. This study reveals an increase in ESBL-producing Enterobacteriaceae among Thai isolates and demonstrates gaps in the current CLSI disk diffusion susceptibility guidelines; it indicates the results of ceftazidime and cefepime disk diffusion susceptibility testing using CLSI criteria should be interpreted with caution.

Aims: In this study, we compared different methods of colistin susceptibility testing, disc diffusion, agar dilution and Etest using a set of Enterobacteriaceae isolates that included colistin-resistant strains., Methods and Results: Susceptibility of 200 clinical isolates of Enterobacteriaceae to colistin was tested to compare agar dilution (reference method), disc diffusion (50 and 10 μg) and Etest. MICs (minimum inhibitory concentrations) were interpreted using the criteria established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Colistin exhibited excellent activity against Escherichia coli and E. cloacae (MIC90 = 0·5 mg l(-1)). In contrast, colistin was less active against Klebsiella pneumoniae (MIC90 = 16 mg l(-1)). Resistance rates varied from 0% in E. coli to 1·8% in E. cloacae and 13% in K. pneumoniae. High rates of very major errors were observed in the disc diffusion test using either the criteria of the Comité de l'antibiogramme de la Société Française de Microbiologie (CA-SFM) or the criteria of the Clinical and Laboratory Standards Institute (CLSI), respectively, 3·5 and 2·5%. When the criteria of Gales et al. were applied, the number of very major errors was reduced to one (0·5%). The Etest showed good concordance with agar dilution method., Conclusion: Disc susceptibility testing methods are unreliable on detecting colistin resistance. MIC should be determined to confirm the susceptibility results by disc diffusion., Significance and Impact of the Study: We recommend the determination of MIC by Etest for all multidrug-resistant Enterobacteriaceae when colistin is required for the treatment., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

Although a variety of techniques are available for antimicrobial susceptibility testing, disk diffusion methods remain the most widely used. We compared the accuracy of disk diffusion zone diameters as obtained by manual zone measurements in a low resource country (Indonesia) to that by automated zone measurements (Oxoid aura image system) in a high resource setting (the Netherlands) to determine susceptibility categories (sensitive, intermediate susceptible or resistant). A total of 683 isolates were studied, including 294 Staphylococcus aureus, 195 Escherichia coli and 194 other Enterobacteriaceae. Antimicrobial agents included tetracycline, oxacillin, gentamicin, erythromycin, trimethoprim/sulfamethoxazole and chloramphenicol for S. aureus and ampicillin, gentamicin, cefotaxime, ciprofloxacin, trimethoprim/sulfamethoxazole, and chloramphenicol for E. coli and other Enterobacteriaceae. Of the 4098 drug-organism combinations, overall category agreement (CA), major discrepancy (MD) and minor discrepancy (mD) between the two methods were 82.4% (3379/4098), 6.0% (244/4098) and 11.6% (475/4098), respectively. One hundred and sixty three of 244 MDs were resolved using reference broth microdilution method. Overall very major error (VME), major error (ME) and minor error (mE) of manual zone measurement were 28.8%, 45.4% and 4.9%, respectively and for the aura image system 4.9%, 16.0% and 4.9%, respectively. The results of this study indicate that the disk diffusion method with manual zone measurement in Indonesia is reliable for susceptibility testing. The use of an automated zone reader, such as the aura image system, will reduce the number of errors, and thus improve the accuracy of susceptibility test results for medically relevant bacteria.

The emergence of metallo-beta-lactamase (MBL)-producing isolates is a challenge to routine microbiology laboratories, since there are no standardized methods for detecting such isolates. The aim of this study was to evaluate the accuracy of different phenotypic methods to detect MBL production among Pseudomonas spp., Acinetobacter spp., and enterobacterial isolates, including GIM, IMP, SIM, SPM, and VIM variants. A total of 46 genetically unrelated Pseudomonas aeruginosa, Pseudomonas putida, Acinetobacter sp., and enterobacterial strains producing distinct MBLs were tested. Nineteen strains were included as negative controls. The inhibition of bacterial growth and beta-lactam hydrolysis caused by MBL inhibitors (IMBL) also were evaluated. The isolates were tested for MBL production by both a double-disk synergy test (DDST) and a combined disk assay (CD) using imipenem and ceftazidime as substrates in combination with distinct IMBL. One hundred percent sensitivity and specificity were achieved by DDST using 2-mercaptopropionic acid in combination with ceftazidime and imipenem for the detection of MBL production among P. aeruginosa and Acinetobacter species isolates, respectively. The CD test showed the same results for detecting MBL-producing enterobacteria by combining imipenem and EDTA, with a 5.0-mm-breakpoint increase in the size of the inhibition zone. Our results indicate that both phenotypic methods to detect MBL-producing isolates should be based on the genera to be tested, regardless of the enzyme produced by such isolates, as well as on the local prevalence of MBL producers.

With the growing frequency of extended-spectrum beta-lactamases (ESBL) among Enterobacteriaceae, treatment of Gram-negative nosocomial infections requires rapid and reliable detection of this enzyme. Quicolor agar (QC agar) (Salubris Inc., Massachusetts, USA) is a novel chromogenic agar medium changing colour within 4 to 6 h due to the metabolic activity of growing bacteria. This study investigated the use of QC agar compared to Mueller Hinton agar (MH) for the detection of ESBL using disk diffusion and E-test. 100 Enterobacteriaceae isolated at Hacettepe University Hospital, of which 50 were predetermined to be ESBL positive and 50 as negative using the CLSI disk diffusion ESBL (phenotypic confirmatory test) criteria. For disk diffusion and E-test, cefotaxime+/-clavulanate (CT/CTL) and ceftazidime+/-clavulanate (TZ/TZL) were used, and for E-test, cefepime+/-clavulanate (PM/PML) was also used. QC agar rapid ESBL results for all strains were in agreement with the standard overnight procedure. All 50 ESBL positives were detected by both methods. For the 50 ESBL negatives, QC agar rapid results from E-test and disk diffusion were in complete accordance with the overnight MH results. Moreover, E-test detected 8 additional ESBL positive strains that disk diffusion missed. For disk diffusion, CT/CTL alone detected all 50 ESBL positives while TZ/TZL alone missed 5 ESBL positives. E-test CT/CTL alone confirmed all 50 ESBL positives and identified 4 additional ESBL-positive strains. When used together, E-test CT/CTL, TZ/TZL and PM/PML identified a total of 58 ESBL positives among the 100 strains tested. QC agar can be used for rapid and reliable ESBL detection within 4 to 6 h, using disk diffusion and E-test ESBL reagents. This rapid method should be further validated using genotype characterized ESBL and other beta-lactamase positive strains.

A prospective study is carried out to evaluate the performance of a protocol for the accelerated detection of extended-spectrum beta-lactamases (ESBLs) in clinical isolates of Escherichia coli, Klebsiella pneumoniae and other Gram-negative bacteria. A modified double-disc test (MDDT) is incorporated in a Gram-negative template for routine susceptibility testing. The MDDT identified accurately ESBLs in all isolates subsequently confirmed as ESBL-producers by the standard Clinical Laboratory Standards Institute (CLSI) combined disc method. Of 1213 isolates tested, 98 (8%) were positive for ESBLs by MDDT and 95 (7.8%) were positive by the CLSI method. ESBLs were detected in 48 (7.8%) E. coli, 21 (8%) K. pneumoniae, 12 (5.8%) Proteus mirabilis, 13 (18.8%) Providencia stuartii and four (6.8%) Enterobacter cloacae isolates. Time required for ESBL detection by the MDDT method was one day. The protocol described provides a simple, rapid and low-cost method for early detection of ESBLs in Gram-negative bacteria.