Novel Disk Diffusion Assay on Magnesium Oxalate Agar To Evaluate the Susceptibility of Yersinia pestis to Type III Secretion System Inhibitors.

Current methods for screening small molecules that inhibit the plasmid pCD1-encoded Yersinia pestis type III secretion system (T3SS) include lengthy growth curves followed by multistep luminescence assays or Western blot assays to detect secretion, or lack thereof, of effector proteins. The goal of this research was to develop a novel disk diffusion assay on magnesium oxalate (MOX) agar as a simple way to evaluate the susceptibility of Y. pestis to type III secretion system inhibitors. MOX agar produces distinct Y. pestis growth characteristics based on the bacteria's ability or inability to secrete effector proteins; small, barely visible colonies are observed when secretion is activated versus larger, readily visible colonies when secretion is inhibited. Wild-type Y. pestis was diluted and spread onto a MOX agar plate. Disks containing 20 µl of various concentrations of imidocarb dipropionate, a known Y. pestis T3SS inhibitor, or distilled water (dH2O) were placed on the plate. After incubation at 37°C for 48 h, visible colonies of Y. pestis were observed surrounding the disks with imidocarb dipropionate, suggesting that T3S was inhibited. The diameter of the growth of colonies surrounding the disks increased as the concentration of the T3SS inhibitor increased. Imidocarb dipropionate was also able to inhibit Y. pestis strains lacking effector Yops and Yop chaperones, suggesting that they are not necessary for T3S inhibition. This disk diffusion assay is a feasible and useful method for testing the susceptibility of Y. pestis to type III secretion system inhibitors and has the potential to be used in a clinical setting. IMPORTANCE Disk diffusion assays have traditionally been used as a simple and effective way to screen compounds for antibacterial activity and to determine the susceptibility of pathogens to antibiotics; however, they are limited to detecting growth inhibition only. Consequently, antimicrobial agents that inhibit virulence factors, but not growth, would not be detected. Therefore, we developed a disk diffusion assay that could detect inhibition of bacterial virulence factors, specifically, type III secretion systems (T3SSs), needle-like structures used by several pathogenic bacteria to inject host cells with effector proteins and cause disease. We demonstrate that magnesium oxalate (MOX) agar can be used in a disk diffusion assay to detect inhibition of the T3SS of Yersinia pestis, the causative agent of bubonic plague, by small-molecule inhibitors. This assay may be useful for screening additional small molecules that target bacterial T3SSs or testing the susceptibility of patient-derived samples to drugs that target T3SSs.

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.

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.

Colistin susceptibility test evaluation of multiple-resistance-level Pseudomonas aeruginosa isolates generated in a morbidostat device.

Objectives: Colistin is a last-resort antibiotic against the critical-status pathogen Pseudomonas aeruginosa. There is still uncertainty regarding how to accurately measure colistin susceptibility in P. aeruginosa. Evaluation of antimicrobial susceptibility testing (AST) methods is largely hampered by the lack of resistant isolates and those around the susceptibility breakpoint. The aim of this study was to generate such strains in a morbidostat device for use in AST method evaluation. Methods: A morbidostat device was used to cultivate susceptible clinical strains into isolates with a wide range of colistin MICs. Subsequently, five commercial AST methods were compared against the gold standard broth microdilution (BMD) method: MICRONAUT-S, SensiTest, Sensititre, Rapid Polymyxin Pseudomonas and Etest. Results: A total of 131 P. aeruginosa isolates were used for colistin susceptibility test evaluation (100 colistin susceptible and 31 colistin resistant). The 31 colistin-resistant isolates evolved resistance in the morbidostat to different MIC ranges (4-512 mg/L, 100% resistance generation efficacy). The categorical agreement (CA) rates for MICRONAUT-S, SensiTest and Rapid Polymyxin Pseudomonas were 94.7%, 93.9% and 92.4%, respectively. The Sensititre achieved the highest CA score (96.9%), whereas the Etests had the lowest CA score (84%). The very major discrepancy (VMD) rates for all tests were between 3.2% and 67.7%. Conclusions: The morbidostat device can efficiently provide laboratories with colistin-resistant strains for test evaluation. Although CA rates were high for commercial AST methods except for Etests, none met the ≤1.5% CLSI limit for VMD rates. Performance was generally inferior when using isolates with low-level resistance.

The Continued Value of Disk Diffusion for Assessing Antimicrobial Susceptibility in Clinical Laboratories: Report from the Clinical and Laboratory Standards Institute Methods Development and Standardization Working Group.

Expedited pathways to antimicrobial agent approval by the U.S. Food and Drug Administration (FDA) have led to increased delays between drug approval and the availability of FDA-cleared antimicrobial susceptibility testing (AST) devices. Antimicrobial disks for use with disk diffusion testing are among the first AST devices available to clinical laboratories. However, many laboratories are reluctant to implement disk diffusion testing for a variety of reasons, including dwindling proficiency with this method, interruptions of the laboratory workflow, uncertainty surrounding the quality and reliability of disk diffusion tests, and a perceived need to report MIC values to clinicians. This minireview provides a report from the Clinical and Laboratory Standards Institute Methods Development and Standardization Working Group on the current standards and clinical utility of disk diffusion testing.

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).

In vitro antifungal susceptibility of clinical isolates of Fusarium from Colombia / Susceptibilidad antifúngica in vitro de aislamientos clínicos de Fusarium de Colombia

ABSTRACT Objective The aim of the present study was to evaluate the antifungal susceptibilities of isolates of Fusarium to amphotericin B, itraconazole and voriconazole. Methods The susceptibility of 44 isolates of Fusarium was tested by the E-test methodology. Results All the isolates were resistant to itraconazole, and 89 % and 54,5 % were resistant to amphotericin B and voriconazole, respectively. Discussion The results confirm the high level of resistance reported, regardless of the species or the strain of Fusarium involved. The high MICs level observed are worrying and suggest that new drugs are needed.(AU)

RESUMEN Objetivo Evaluar la susceptibilidad antifúngica in vitro de aislamientos de Fusarium a los antimicóticos amfotericina B, itraconazol y voriconazol. Métodos La susceptibilidad de 44 aislamientos clínicos de Fusarium fue evaluada por el método de difusión en disco, E-test. Resultados Todos los aislamientos fueron resistentes al itraconazol, y 89 % y 54,5 % fueron resistentes a la amfotericina B y al voriconazol, respectivamente. Discusión Los resultados confirman el alto nivel de resistencia reportado, independiente de la especie o la cepa de Fusarium involucrada. Los valores tan altos de MICs son preocupantes y sugieren la necesidad de evaluar nuevos medicamentos.(AU)

Fully automated disc diffusion for rapid antibiotic susceptibility test results: a proof-of-principle study.

Background: Antibiotic resistance poses a significant threat to patients suffering from infectious diseases. Early readings of antibiotic susceptibility test (AST) results could be of critical importance to ensure adequate treatment. Disc diffusion is a well-standardized, established and cost-efficient AST procedure; however, its use in the clinical laboratory is hampered by the many manual steps involved, and an incubation time of 16-18 h, which is required to achieve reliable test results. Methods: We have evaluated a fully automated system for its potential for early reading of disc diffusion diameters after 6-12 h of incubation. We assessed availability of results, methodological precision, categorical agreement and interpretation errors as compared with an 18 h standard. In total, 1028 clinical strains (291 Escherichia coli , 272 Klebsiella pneumoniae , 176 Staphylococcus aureus and 289 Staphylococcus epidermidis ) were included in this study. Disc diffusion plates were streaked, incubated and imaged using the WASPLab TM automation system. Results and conclusions: Our results demonstrate that: (i) early AST reading is possible for important pathogens; (ii) methodological precision is not hampered at early timepoints; and (iii) species-specific reading times must be selected. As inhibition zone diameters change over time and are phenotype/drug combination dependent, specific cut-offs and expert rules will be essential to ensure reliable interpretation and reporting of early susceptibility testing results.

Validation of novel cost-effective antimicrobial gradient strips for the determination of carbapenem susceptibility in Enterobacteriaceae.

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 log2 dilution) with the reference methods of agar dilution, BMD and Etest, respectively; furthermore, essential agreement was >90% within ±2 log2 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.

Evaluation of the PREVI® Isola automated seeder system compared to reference manual inoculation for antibiotic susceptibility testing by the disk diffusion method.

The disk diffusion (DD) method remains the most popular manual technique for antibiotic susceptibility testing (AST) in clinical microbiology laboratories. This is because of its simplicity, reproducibility, and limited cost compared to (automated) microdilution systems, which are usually less sensitive at detecting certain important mechanisms of resistance. Here, we evaluate the PREVI® Isola automated seeder system using a new protocol for spreading bacterial suspensions (eight deposits of calibrated inocula of bacteria, followed by two rounds of rotation) in comparison with manual DD reference testing on a large series of clinical and reference strains. The average time required for seeding one agar plate for DD with this new protocol was 51 s per plate, i.e., 70 agar plates/h. Reproducibility and repeatability was assessed on three reference and three randomly chosen clinical strains, as usually requested by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and was excellent compared to the manual method. The standard deviations of zones of growth inhibition showed no statistical discrimination. The correlation between the two methods, assessed using 294 clinical isolates and a panel of six antibiotics (n = 3,528 zones of growth inhibition measured), was excellent, with a correlation coefficient of 0.977. The new PREVI® Isola protocol adapted for DD had a sensitivity of 99 % and a specificity of 100 % compared to the manual technique for interpreting DD as recommended by the EUCAST.

Antimicrobial susceptibility assays in paper-based portable culture devices.

To detect antibiotic-resistant bacteria in areas remote from microbiology laboratories, we designed portable culture devices performing an analogue of the Kirby-Bauer disk diffusion test inside patterned papers embedded in tape. We quantified the antibiotic susceptibility of several strains of Escherichia coli and Salmonella typhimurium by measuring blue-colored zones of inhibited growth.

Comparison of Rosco Neo-Sensitabs with Oxoid paper disks in EUCAST disk diffusion antimicrobial susceptibility testing on Mueller-Hinton agar.

This study compared Neo-Sensitabs with Oxoid paper disks using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) disk diffusion antimicrobial susceptibility test on Mueller-Hinton agar. The EUCAST-recommended quality control strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212) (Part I) and clinical isolates (Part II) were investigated. In Part I of the study, 27 combinations of antimicrobial agents were tested on four quality control strains repeatedly up to 60 times and zone diameters of tablets and disks were compared. In Part II of the study, 351 clinical isolates were included to cover a broad range of species, as well as resistance mechanisms. In Part I, four major deviations (>1 mm outside quality control ranges) were observed with Neo-Sensitabs. In one case with P. aeruginosa ATCC 27853 (meropenem), there was a corresponding major deviation (2 mm) with the Oxoid disk. The three remaining major deviations with Neo-Sensitabs were observed with meropenem (2 mm) in E. coli ATCC 25922 and with ciprofloxacin (2 mm) and gentamicin (3 mm) in P. aeruginosa ATCC 27853. For Oxoid disks, there were only minor deviations (=1 mm outside quality control ranges) in these three cases. In Part II, there were six discrepancies, susceptible versus resistant, in 3,533 comparisons between the two methods with the clinical isolates. The Rosco Neo-Sensitabs appear to be a possible alternative to Oxoid paper disks for EUCAST disk diffusion antimicrobial susceptibility testing on Mueller-Hinton agar.

Frequency and antibiogram of multi-drug resistant Pseudomonas aeruginosa.

OBJECTIVE: To find out the frequency and susceptibility pattern of multi-drug resistant (MDR) Pseudomonas aeruginosa in clinical specimens. STUDY DESIGN: Cross-sectional observational study. PLACE AND DURATION OF STUDY: Department of Microbiology, Army Medical College, National University of Sciences and Technology (NUST), Rawalpindi, from January to September 2010. METHODOLOGY: Routine clinical specimens were subjected to standard microbiological procedures and the isolates were identified to the species level. The antibiotics susceptibility was determined by Kirby Bauer Disc diffusion method and the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: The frequency of MDR Pseudomonas aeruginosa among all the Pseudomonas aeruginosa strains isolated was found to be 22.7%. These isolates were most sensitive to Colistin followed by Piperacillin-Tazobactam and Cefoperazone-Sulbactum. CONCLUSION: Increasing fequency of infections due to MDR Pseudomonas aeruginosa is an emerging threat in our set up which an be prevented by prescribing antibiotics judiciously and by adopting proper disinfection measures.

Comparison of disk diffusion, Etest and VITEK2 for detection of carbapenemase-producing Klebsiella pneumoniae with the EUCAST and CLSI breakpoint systems.

The aim of this study was to compare CLSI and EUCAST MIC and disk diffusion carbapenem breakpoints for the detection of carbapenemase-producing Klebsiella pneumoniae. K. pneumoniae strains with known KPC (n = 31) or VIM (n = 20) carbapenemases were characterized by disk diffusion (Oxoid) and Etest (bioMérieux) vs. imipenem, meropenem and ertapenem, and with VITEK2 (bioMérieux, five different cards). Extended-spectrum ß-lactamase (ESBL) testing was performed with VITEK2 (bioMérieux), ESBL combination disks (Becton Dickinson) and the ESBL Etest (bioMérieux). With CLSI and EUCAST MIC breakpoints, respectively, 11 and seven of the strains were susceptible to imipenem, 12 and eight to meropenem, and seven and none to ertapenem. The EUCAST epidemiological cut-off (ECOFF) values for meropenem and ertapenem identified all carbapenemase producers, whereas the imipenem ECOFF failed in five strains. All carbapenemase producers were detected with EUCAST disk diffusion breakpoints for ertapenem and meropenem, and four strains were susceptible to imipenem. CLSI disk diffusion breakpoints characterized 18 (imipenem), 14 (meropenem) and three (ertapenem) isolates as susceptible. When cards with a single carbapenem were used, detection failures with VITEK2 were four for imipenem, none for meropenem and one for ertapenem. Cards containing all three carbapenems had one to two failures. With ESBL combination disks, 21/31 KPC producers and 2/20 VIM producers were positive. With VITEK2, no VIM producers and between none and seven KPC producers were ESBL-positive. All carbapenemase producers were detected with the meropenem MIC ECOFF, or the clinical EUCAST breakpoint for ertapenem. EUCAST disk diffusion breakpoints for meropenem and ertapenem detected all carbapenemase producers. VITEK2 had between none and four failures in detecting carbapenemase producers, depending on the antibiotic card.

[Evaluation and utility of the E-test and Neo-Sensitabs methods in studying fluconazole yeast susceptibility]. / Evaluacion y utilidad de los metodos E-test y Neo-Sensitabs para estudiar la sensibilidad de las levaduras al fluconazol.

Standardized broth dilution methods are cumbersome for routine use in a clinical laboratory to study antifungal yeast susceptibility. Recently, the CLSI has standardized a disk diffusion method faster and more suitable to study fluconazole and voriconazole susceptibility. The objectives of the present study were to determine: a) the suitability of the Neo-Sensitabs tablets to study fluconazole susceptibility; b) whether Mueller-Hinton agar with methylene blue (MHAG-AM) could be used in the E-test method; and c) the interaction of the methylene blue with RPMI medium. A total of 84 blood stream yeast isolates were used (25 C. albicans, 7 C. parapsilosis, 10 C. tropicalis, 12 C. glabrata, 7 C. krusei, 4 C. lusitaniae and 19 C. neoformans). The methylene blue makes sharper inhibition zones both in MHAG-AM and RPMI media. With fluconazole Neo-Sensitabs tablets, the lowest percentage of very major errors was found in MHAG-AM and the greatest in RPMIG. In both diffusion methods and culture media, the very major errors were found in C. albicans, C. tropicalis (only with Neo-Sensitabs) and C. glabrata. The percentage of fluconazole-resistant strains was lower in the media that contained glucose (2%). Neo-Sensitabs tablets are a reliable alternative to the dilution methods to detect fluconazole susceptibility. In the case of resistance, more studies are required; nevertheless, inhibition zone > or =17 mm should be applied to define fluconazole resistance.

"All-in-one-plate" E-test and disk diffusion susceptibility co-testing for multiresistant Acinetobacter baumannii.

Multiresistant Acinetobacter baumannii requires ancillary manual susceptibility testing with the E-test and disk diffusion when tested with the VITEK-2 system (bioMérieux, Marcy l'Etoile, France). In the study presented here, the E-test and disk diffusion were combined in a single plate, and the method was verified by comparing categorical agreement of combined and control plates. There were no very major, major or minor errors, and similar results were obtained for all ten representative bacterial strains used as controls. Co-testing is thus feasible, accurate and reproducible, and it merits evaluation with other bacterial species.