Commercial Broth Microdilution Research Articles

Comparison of two commercial broth microdilution panels for multidrug-resistant Gram-negative bacteria: Thermo Scientific™ Sensititre DKMGN vs. Beckman Coulter MicroScan NMDRM1.

Antimicrobial susceptibility testing (AST) using broth microdilution (BMD) is usually the reference method to obtain accurate minimum inhibitory concentrations and optimally manage infections with resistant organisms. Several commercial dry BMD are available for AST in clinical laboratories. Two commercial BMD panels for testing of multidrug-resistant Gram-negative bacteria were compared: the Thermo Scientific™ Sensititre DKMGN and the Beckman Coulter NMDRM1, for 17 antimicrobial agents. A total of 207 isolates were tested: three ATCC strains and one NCTC strain, six quality control strains from the Belgian National Antimicrobial Committee, and 197 clinical isolates, including carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2023 breakpoints version 13.1 were used to assign susceptibility categories. Overall, the categorical agreement (CA) and essential agreement (EA) were both above 90%, but several useful antibiotics for the treatment of multi-resistant organisms showed CA and EA under 90%, that is, meropenem, imipenem, and colistin for Enterobacterales and meropenem and colistin for P. aeruginosa. For Enterobacterales, the NMDRM1 panel showed a significantly higher resistance rate for meropenem, imipenem, amikacin, and colistin. For carbapenems, the minimal inhibitory concentrations (MICs) were underestimated by the DKMGN panel, as already pointed out by a warning on the EUCAST website. To better assess carbapenem susceptibility in carbapenem-resistant organisms, the DKMGN panel now requires the use of a higher inoculum in the insert kit. However, for a given isolate whose susceptibility to carbapenems is not known, there is a risk of underestimating the MIC values. Our results show that colistin testing remains a challenge, highlighting the urgent need for the development of more accurate commercial methods. The use of a single commercial method cannot guarantee good precision in the determination of the MIC value for colistin.

Evaluation of the Colistin HiMIC Plate Kit for Colistin Susceptibility Testing of Klebsiella pneumoniae.

Colistin HiMIC Plate Kit (HiMedia Laboratories), a new commercial broth microdilution (BMD) test for colistin susceptibility testing was evaluated. BMD according to ISO standard 20776-1 (2019) with two-fold dilutions from 128 to 0.125 mg/L was used as a reference method. The colistin reference MICs (minimal inhibitory concentration) ranged from 0,25 to 128 mg/L with 15 (20.5%; 15/73) isolates having colistin reference MICs close to the current EUCAST breakpoint (MICs of 2, 4, and 8 mg/L). The study assessed the compliance of a commercial kit with the CLSI criteria, including categorical agreement (CA) and essential agreement (EA ≥90%), very major error (VME rate) <3%, and major error (ME) rate <3%. On 73 carbapenemase-producing Klebsiella pneumoniae isolates Colistin HiMICTM Plate Kit showed CA and EA of 100% (73/73; 95% CI 0.97-1.00) and 82.2% (60/73; 95% CI: 0.72-0.90), respectively. No ME (false-resistant results) and VME (false-susceptible results) were detected. Kit showed acceptable CA, ME, and VME error parameters, whereas the EA did not meet the ≥90% threshold. Laboratories must check for possible limitations of commercial kits before they can be used for colistin susceptibility testing.

Real-world performance of susceptibility testing for cefiderocol: insights from a prospective multicentre study on Gram-negative bacteria.

Cefiderocol is a novel siderophore-conjugated cephalosporin developed for the treatment of multidrug-resistant Gram-negative bacterial (GNB) infections. However, the current gold standard for cefiderocol susceptibility testing, broth microdilution (BMD) using iron-depleted cation-adjusted Mueller-Hinton broth, presents challenges for many microbiology laboratories. In this study, we evaluate the real-world performance of disc diffusion (DD) and a commercial BMD method (ComASP®) to test cefiderocol susceptibility in a series of isolates collected prospectively from severely ill patients in a multicentre study. The susceptibilities of 1472 isolates (632 Enterobacterales, 532 Pseudomonas aeruginosa, 84 Acinetobacter spp. and 224 Stenotrophomonas maltophilia) collected in 60 Spanish hospitals were analysed following the EUCAST 2023 and 2024 criteria. We assessed the performance of DD (cefiderocol 30 μg disc, Liofilchem) and a commercial BMD method (ComASP® Cefiderocol, Liofilchem). A total of 1408 and 1450 isolates were susceptible by DD and ComASP® BMD, respectively. Overall, the agreement between both methods was 96.9%. Forty-four isolates were resistant by DD but susceptible by ComASP® BMD, and two were susceptible by DD but resistant by ComASP® BMD (Acinetobacter baumannii isolates). Adoption of the updated 2024 EUCAST DD breakpoints and areas of technical uncertainty (ATUs) led to a decrease in susceptibility among Enterobacterales (95.3% versus 92.6%). DD is a straightforward, rapid and accessible method for routine determination of cefiderocol susceptibility in real-world practice. ComASP® BMD shows a high agreement with DD in susceptible isolates and may help to resolve DD interpretability concerns in isolates with susceptibility results within the ATU, but caution is warranted when testing resistant isolates.

Evaluation of two commercial broth microdilution systems for dalbavancin susceptibility testing of methicillin-resistant Staphylococcus aureus and other resistant Gram-positive cocci

ObjectivesThis study aims to evaluate two commercial broth microdilution (BMD) systems, E1-185-100 (Merlin) and FDANDPF (ThermoFisher), for dalbavancin susceptibility testing in comparison with reference BMD assay. MethodsStudy collection was composed of 200 non-replicate multidrug-resistant Gram-positive cocci of clinical origin, including 180 methicillin-resistant Staphylococcus aureus, 10 vancomycin-resistant enterococci, seven linezolid-resistant Staphylococcus epidermidis, and three methicillin-resistant coagulase-negative staphylococci. S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 reference strains were also included as controls. Testing was performed according to the ISO 20776-1 standard, starting from the same bacterial inoculum, and results were compared according to the ISO 20776-2 standard. ResultsReference BMD showed that 92.6% (187/202) of the strains were susceptible to dalbavancin, whereas few staphylococci and all VanA-producing enterococci showed a resistant phenotype. In comparison with the reference method, Category Agreement and Essential Agreement were 98% (198/202; 95% CI, 95.4–99.3%) and 98% (198/202; 95% CI, 95.4–99.3%) for both Merlin and ThermoFisher panels. A few false susceptibilities were observed, for both commercial systems, with dalbavancin-resistant staphylococci. BIAS values of 11% and 3% were calculated for the Merlin and ThermoFisher systems, respectively. DiscussionThis study, reporting the first evaluation of the two commercially available BMD assays for dalbavancin susceptibility testing, revealed an overall good correlation with reference BMD, although with some underestimation tendency of MIC values by both commercial systems. Further studies involving a higher number of resistant isolates will be necessary to better evaluate this issue.

Comparison of Minimum Inhibitory Concentrations of Selected Antimicrobials for Non-Aureus Staphylococci, Enterococci, Lactococci, and Streptococci Isolated from Milk Samples of Cows with Clinical Mastitis.

The objective of this study was to compare the minimum inhibitory concentrations of antimicrobials included in a commercial broth microdilution panel among Gram-positive pathogens that caused non-severe clinical mastitis on three Michigan dairy farms. Duplicate quarter milk samples were collected from eligible quarters of cows enrolled in a randomized clinical trial, cultured in a university laboratory, and identified using MALDI-TOF. Etiologies were grouped by genus as Enterococcus species (n = 11), Lactococcus species (n = 44), non-aureus Staphylococcus species (n = 39), or Streptococcus species (n = 25). Minimum inhibitory concentrations (MICs) were determined using the mastitis panel of a commercially available broth microdilution test. In vitro susceptibility was determined using approved guidelines and included breakpoints for mastitis pathogens, or when not available, breakpoints from other species. Most isolates were inhibited at or below breakpoints that demonstrated in vitro susceptibility. The proportions of susceptible isolates varied among pathogens for pirlimycin, penicillin, and tetracycline. The greatest proportion of resistance was observed for pirlimycin, tetracycline, and sulfadimethoxine. Survival analysis was performed to evaluate differences in MICs among pathogen groups. MIC values varied among pathogens for ceftiofur, cephalothin, erythromycin, penicillin, pirlimycin, and tetracycline. However, nearly all isolates were susceptible to ceftiofur and cephalothin, indicating that pathogen differences in MIC are not likely clinically relevant, as these are the two most commonly administered mastitis treatments in the United States. While differences in vitro susceptibility were observed for some antimicrobials, susceptibility was high to cephalosporin-based IMM treatments that are most commonly used and did not vary among pathogens.

Proficiency testing for drug susceptibility testing of Mycobacterium tuberculosis complex using commercial broth microdilution plate in China in 2021

The characteristic and performance of Broth microdilution (BMD) plates for drug susceptibility of Mycobacterium tuberculosis have not been systematically evaluated in China. This study was designed to review the key information and assess the performance of BMD plates by analysis of proficiency testing results. We retrospectively analysed the proficiency testing results of phenotypic drug susceptibility testing (PT-DST) of 45 laboratories using BMD plates in China in 2021. Critical information, such as drug layout, concentration range of each drug, plate storage conditions and duration, operating procedures, and interpretation criteria for binary results were compared. The performance was also analysed. Eight types of BMD plates produced by four manufactures were reported. The drug layout, number of drugs on plates, and concentration range varied a lot between different plates. The total sensitivity and specificity of BMD plates for drug susceptibility of Mycobacterium tuberculosis to ten drugs (isoniazid (INH), rifampin (RIF), kanamycin (KAM), amikacin (AM), levofloxacin (LFX), moxifloxacin (MFX), bedaquiline (BDQ), linezolid (LZD), clofazimine (CFZ), and delamanid (DLM)) were 93.9% (95% CI 92.-94.9) and 99.1% (95% CI 98.8-99.3), respectively. The lowest sensitivity was 84.8% (95% CI 80.3-88.4) for LFX and 86.4% (95% CI 82.5-89.6) for MFX, or 87.5% (95% CI 84.2-90.2) for Y1 plate and 87.9% (95% CI 83.5-91.1) for T plate. The lowest specificity was 94.4% (95% CI 91.4-96.4) for DLM, or 97.9% (95% CI 96.8-98.7) for B3 plate. Commercial BMD plates in China showed varied drug layouts and operational procedures, indicating the urgency of standardization. The lower performance for some drugs showed the low quality of the plates utilized or lack of proficiency of lab staffs in operating and interpreting results.

Antimicrobial Resistance Laboratory Network's multisite evaluation of the ThermoFisher Sensititre GN7F broth microdilution panel for antimicrobial susceptibility testing.

In 2017, the Centers for Disease Control and Prevention (CDC) established the Antimicrobial Resistance Laboratory Network to improve domestic detection of multidrug-resistant organisms. CDC and four laboratories evaluated a commercial broth microdilution panel. Antimicrobial susceptibility testing using the Sensititre GN7F (ThermoFisher Scientific, Lenexa, KS) was evaluated by testing 100 CDC and Food and Drug Administration AR Isolate Bank isolates [40 Enterobacterales (ENT), 30 Pseudomonas aeruginosa (PSA), and 30 Acinetobacter baumannii (ACB)]. We assessed multiple amounts of transfer volume (TV) between the inoculum and tubed 11-mL cation-adjusted Mueller-Hinton broth: 1 µL [tribe Proteeae (P-tribe) only] and 10, 30, and 50 µL, resulting in respective CFU per milliter of 1 × 104, 1 × 105, 3 × 105, and 5 × 105. Four TV combinations were analyzed: standard (STD) [1 µL (P-tribe) and 10 µL], enhanced standard (E-STD) [1 µL (P-tribe) and 30 µL], 30 µL, and 50 µL. Essential agreement (EA), categorical agreement, major error (ME), and very major error (VME) were analyzed by organism then TVs. For ENT, the average EA across laboratories was <90% for 7 of 15 β-lactams using STD and E-STD TVs. As TVs increased, EA increased (>90%), and VMEs decreased. For PSA, EA improved as TVs increased; however, MEs also increased. For ACB, increased TVs provided slight EA improvements; all TVs yielded multiple VMEs and MEs. For ENT and ACB, Minimum inhibitory concentrations (MICs) trended downward using a 1 or 10 µL TV; there were no obvious MIC trends by TV for PSA. The public health and clinical consequences of missing resistance warrant increased TV of 30 µL for the GN7F, particularly for P-tribe, despite being considered "off-label" use.

Correlation of drug exposure and bacterial susceptibility with treatment response for Mycobacterium avium complex lung disease: protocol for a prospective observational cohort study

IntroductionThe burden of Mycobacterium avium complex (MAC) lung disease is increasing globally and treatment outcome is in general poor. Therapeutic drug monitoring has the potential to improve treatment outcome by...

Comparison of Disk Diffusion, E-Test, and Broth Microdilution Methods for Testing In Vitro Activity of Cefiderocol in Acinetobacter baumannii.

The reference method for cefiderocol antimicrobial susceptibility testing is broth microdilution (BMD) with iron-depleted-Mueller-Hinton (ID-MH) medium, whereas breakpoints recommended for disk diffusion (DD) are based on MH-agar plates. We aimed to compare the performance of the commercial BMD tests ComASP (Liofilchem) and UMIC (Bruker), and DD and E-test using MH- and ID-MH-agar plates with the reference BMD method using 100 carbapenem-resistant-A. baumannii isolates. Standard BMD was performed according to the EUCAST guidelines; DD and E-test were carried out using two commercial MH-agar plates (BioMérieux and Liofilchem) and an in-house ID-MH-agar plate, while ComASP and UMIC were performed according to the manufacturer's guidelines. DD performed with the ID-MH-agar plates led to a higher categorical agreement (CA, 95.1%) with standard BMD and fewer categorization errors compared to the commercial MH-agar plates (CA BioMérieux 91.1%, Liofilchem 89.2%). E-test on ID-MH-agar plates exhibited a significantly higher essential agreement (EA, 75%) with standard BMD compared to the two MH-agar plates (EA BioMérieux 57%, Liofilchem 44%), and showed a higher performance in detecting high-level resistance than ComASP and UMIC (mean log2 difference with standard BMD for resistant isolates of 0.5, 2.83, and 2.08, respectively). In conclusion, DD and E-test on ID-MH-agar plates exhibit a higher diagnostic performance than on MH-agar plates and the commercial BMD methods. Therefore, we recommend using ID-MH-agar plates for cefiderocol susceptibility testing of A. baumannii.

Towards clinical breakpoints for non-tuberculous mycobacteria – Determination of epidemiological cut off values for the Mycobacterium avium complex and Mycobacterium abscessus using broth microdilution

ObjectiveFor non-tuberculous mycobacteria (NTM), minimum inhibitory concentration (MIC) distributions of wild-type isolates have not been systematically evaluated despite their importance for establishing antimicrobial susceptibility testing (AST) breakpoints. MethodsWe gathered MIC distributions for drugs used against the Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) obtained by commercial broth microdilution (SLOMYCOI and RAPMYCOI) from 12 laboratories. Epidemiological cut-off values (ECOFFs) and tentative ECOFFs (TECOFFs) were determined by EUCAST methodology including quality control (QC) strains. ResultsThe clarithromycin ECOFF was 16 mg/L for M. avium (n = 1271) whereas TECOFFs were 8 mg/L for M. intracellulare (n = 415) and 1 mg/L for MAB (n = 1014) confirmed by analysing MAB subspecies without inducible macrolide resistance (n = 235). For amikacin, the ECOFFs were 64 mg/L for MAC and MAB. For moxifloxacin, the WT spanned >8 mg/L for both MAC and MAB. For linezolid, the ECOFF and TECOFF were 64 mg/L for M. avium and M. intracellulare, respectively. Current CLSI breakpoints for amikacin (16 mg/L), moxifloxacin (1 mg/L) and linezolid (8 mg/L) divided the corresponding WT distributions. For QC M. avium and M. peregrinum, ≥95% of MIC values were well within recommended QC ranges. ConclusionAs a first step towards clinical breakpoints for NTM, (T)ECOFFs were defined for several antimicrobials against MAC and MAB. Broad wild-type MIC distributions indicate a need for further method refinement which is now under development within the EUCAST subcommittee for anti-mycobacterial drug susceptibility testing. In addition, we showed that several CLSI NTM breakpoints are not consistent in relation to the (T)ECOFFs.

Analysis of colistin resistance in carbapenem-resistant Enterobacterales and XDR Klebsiella pneumoniae.

Introduction:Increasing occurrence of infections caused by multidrug-resistant Gram-negative bacteria resulted in colistin being the last agent for treatment. Apart from plasmid-mediated mcr genes, mutations involving several genes like mgrB, phoP/phoQ, pmrA, pmrB, pmrC, and crrABC genes, are leading causes of colistin resistance. Four colistin susceptibility testing methods were compared against broth microdilution (BMD) and determined the presence of the mcr1-5 gene.Methodology:A total of 100 carbapenem-resistant Enterobacterales isolates were tested for colistin susceptibility by commercial broth microdilution (cBMD), E-test, VITEK-2, and rapid polymyxin NP assay (RPNP) and compared with BMD. The presence of the mcr1-5 gene was determined by modified RPNP and PCR. Two non-mcr colistin-resistant XDR isolates were subjected to whole-genome sequencing using Illumina MiSeq sequencing platform.Results:Among 100 carbapenem-resistant Enterobacterales isolates, 15% were resistant to colistin. Essential agreement, categorical agreement, major error, and very major error for cBMD/E-test/VITEK-2/RPNP were 96%/73%/82%/NA; 99%/86%/88%/91%, 1.2%/9.4%/11.8%/8.2% and 0%/40%/13.3%/13.3%, respectively. Only one Klebsiella pneumoniae isolate harbored the mcr-1 gene, observed by both methods. Whole-genome sequencing of two non-mcr XDR Klebsiella pneumoniae showed multiple mutations in 10 genes responsible for lipopolysaccharide biosynthesis.Conclusions:The performance of cBMD was excellent, whereas the E-test was unacceptable. VITEK-2 and RPNP performed better but remained unreliable due to high error rates. Multiple mutations in the target proteins involving lipopolysaccharide formation, modification, and regulation were seen, resulting in colistin resistance.

DETECTION OF COLISTIN RESISTANCE IN MULTIDRUG-RESISTANT GRAM-NEGATIVE BACILLI: PERFORMANCE OF THE BD PHOENIX AUTOMATED SYSTEM VERSUS THE BROTH MICRODILUTION METHOD

Introduction: The increased dissemination of multidrug-resistant Gram-negative bacilli along with a lag in the development of novel, broad-spectrum antimicrobials determined the reintroduction of colistin into clinical practice. In this context, antibiotic susceptibility testing for colistin using high-performance methods has become a requirement. Objective: We compared the performances of the Becton Dickinson (BD) Phoenix 50 automated system and the Micronaut MIC-Strip, a commercial broth microdilution (BMD) method, for the detection of colistin resistance in clinical multidrug-resistant Gram-negative bacilli, isolated from patients admitted in a tertiary hospital in southern Romania. Methods: 54 clinical isolates with different multidrug-resistant phenotypes (Enterobacterales and Pseudomonas aeruginosa) were tested for colistin susceptibility with the BD Phoenix 50 automated system and the Micronaut MIC-Strip, following the Clinical Laboratory Standard Institute recommendations (CLSI, 2021) and were further classified according to the interpretative categories (resistant and intermediate, respectively). The statistical data was calculated using Microsoft Excel 2007 and OpenEPI software. Results: BD Phoenix system failed to detect 11 colistin resistant isolates (false susceptibility). By contrast, all 38 clinical isolates defined as intermediate by the BMD method were also classified as intermediate by the Phoenix system. Conclusion: BD Phoenix system is a reliable technology for detecting colistin resistance. However, high rates of false susceptibility were observed for the BD Phoenix system, indicating that the intermediate / susceptible results should be confirmed with the standard BMD method. Keywords: colistin susceptibility testing, Gram-negative bacilli, multidrug resistance.

Broth Microdilution and Gradient Diffusion Strips vs. Reference Agar Dilution Method: First Evaluation for Clostridiales Species Antimicrobial Susceptibility Testing.

Antimicrobial susceptibility testing of anaerobes is challenging. Because MIC determination is recommended by both CLSI and EUCAST, commercial broth microdilution and diffusion strip tests have been developed. The reliability of broth microdilution methods has not been assessed yet using the agar dilution reference method. In this work, we evaluated two broth microdilution kits (MICRONAUT-S Anaerobes® MIC and Sensititre Anaerobe MIC®) and one gradient diffusion strip method (Liofilchem®) for antimicrobial susceptibility testing of 47 Clostridiales isolates (Clostridium, Clostridioides and Hungatella species) using the agar dilution method as a reference. The evaluation focused on comparing six antimicrobial molecules available in both microdilution kits. Analytical performances were evaluated according to the Food and Drug Administration (FDA) recommendations. Essential agreements (EA) and categorical agreements (CA) varied greatly according to the molecule and the evaluated method. Vancomycin had values of essential and categorical agreements above 90% for the three methods. The CA fulfilled the FDA criteria for three major molecules in the treatment of Gram-positive anaerobic infections (metronidazole, piperacillin/tazobactam and vancomycin). The highest rate of error was observed for clindamycin. Multicenter studies are needed to further validate these results.

Evaluation of the MICRONAUT MIC-strip colistin assay for colistin susceptibility testing of carbapenem-resistant Acinetobacter baumannii and Enterobacterales

We soughtto evaluate the MICRONAUT MIC-Strip colistin (MMS) assay, a commercial broth microdilution (BMD) panel, using 273 carbapenem-resistant Acinetobacter baumannii and Enterobacterales isolates. BMD was used as gold standard. MMS had 98.5% sensitivity and 99.5% specificity. All 37 isolates with MICs close to the breakpoint exhibited complete agreement.

Evaluation of a Commercial Broth Microdilution Panel for Colistin Susceptibility Testing of Clinical Isolates of Escherichia coli and Klebsiella pneumoniae.

Colistin is among the last resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. Antimicrobial susceptibility testing of colistin is challenging due to its physicochemical properties. Broth microdilution (BMD) is the recommended method for colistin susceptibility testing. However BMD is not practical for clinical microbiology laboratories as manual preparation of BMD plates is time-consuming and labor intensive. Recently, some more user-friendly BMD products with commercial panels have become available. Our objective was to evaluate the performance of a commercial broth microdilution (BMD) product [Sensititre (Thermo Fisher Scientific)] for colistin MIC determination by comparison with reference BMD method using a collection of E. coli and K. pneumoniae isolates. A total of 323 unique patient isolates (102 E. coli, 221 K. pneumoniae) were included in the study. Isolates were stored at -70°C and subcultured twice on sheep blood agar before testing. Colistin MICs of the isolates were determined using Sensititre (a premade BMD product with dried antibiotics) and an 'in-house prepared BMD panel prepared in accordance with CLSI guidelines' (reference method). MIC determination with Sensititre was performed according to manufacturer's instructions. The reference method was performed using untreated 96-well sterile polystyrene plates. Colistin MIC results were interpreted according to EUCAST breakpoints (susceptible, ≤ 2 mg/L; resistant, > 2 mg/L). Overall susceptibility rate of isolates to colistin by reference BMD was 75.9%. Overall categorical agreement (CA), essential agreement (EA), very major error (VME), and major error (ME) rates for Sensititre were 98.5%, 72.5%, 3.8%, and 0.8%, respectively. The CA and EA between Sensititre and reference BMD for the isolates with reference colistin MICs close to the susceptibility breakpoint (2 - 8 mg/L) was 94.2% and 48.1%, respectively. Sensititre yielded a VME rate of 15% and ME rate of 0%, respectively, for this subset of isolates. In conclusion, Sensititre showed high CA but low EA with reference BMD for entire collection of isolates. The VME rate was just slightly above 3% and ME rate was acceptable. The rates of CA and EA were decreased and the rate of VME was increased when a subset consisting of more challenging isolates was used.

In-house pre-prepared broth microdilution plates: A simple, cheap and pragmatic approach for susceptibility testing of colistin

Background: With the upsurge in multi-drug resistant Gram-negative bacilli, it is imperative to optimize and standardize the antibiotic susceptibility testing (AST) methods for polymyxins (colistin & polymyxin B). The long overdue confusion regarding the susceptibility testing of colistin was put to rest in 2016 when the CLSI-EUCAST joint Polymyxin Breakpoint Working Group recommended broth microdilution (BMD) in polystyrene plates as the reference method for AST. Though several commercially-available BMD plates have been assessed by EUCAST, the high cost of such commercial plates during contemporary times hinder their use in routine microbiological laboratories especially in low and middle income countries. Thus, a suitable alternative to commercial BMD plates should be sought for to ensure a cost-effective, rapid and reliable reporting of AST of colistin. Methods and materials: With the aim of reducing this redundancy in the workflow, we designed an experiment to charge several microtiter plates with the drug dilution and the media on day 0, storing them frozen at −200C and then retrieving them sequentially for AST (henceforth called pre-prepared frozen BMD plates (PFBP). The PFBP were charged with same representative organisms on every 2–4 days for two months to check for the reliability of AST reporting. Essential agreement (EA), categorical agreement (CA), very major error (VME) and major errors (ME) were computed in accordance with ISO standard 20776-2. For validation, the criteria of EA3 90%, CA 3 90%, VME £1.5% AND ME£3% was used. Pearson correlation and Cohen's kappa (k) score was calculated using GraphPad (San Diego, CA, USA). Results: For colistin, EA and CA of PFBP was 97.5% and 98.3%, respectively. For polymyxin B, EA and CA of PFBP was 99.1% and 90%. Further, the performance of PFBP for colistin susceptibility testing of A. baumannii in the present study (EA=97.5%, CA=98.3%) was superior to five commercial plates available and evaluated by EUCAST. Conclusion: Based on these observations, we propose that pre-prepared frozen BMD plates can be incorporated in routine workflow of microbiological laboratory as reliable, cheap and rapid method for susceptibility testing of colistin.

The accuracy of four commercial broth microdilution tests in the determination of the minimum inhibitory concentration of colistin

Colistin is considered as one of the last-resort antibiotics and reliable antimicrobial susceptibility testing is therefore crucial. The reference standard for AST according to EUCAST and CLSI is broth microdilution (BMD). However, BMD is labor intensive to perform. Commercial antimicrobial susceptibility tests derived from BMD method are available. We investigated the performance of four different commercial tests: Sensititre™, SensiTest™ Colistin, Micronaut MIC Strip Colistin and UMIC Colistin using 70 clinical isolates (half of them was deemed by VITEK2 as resistant), including isolates from cystic fibrosis patients and mcr-1 bearing isolates. We used two reference standards: BMD and composite MIC as determined by all four tests. Sensititre™ had essential agreement (EA, defined as minimum inhibitory concentration within ± 1 dilution) of 87% and 89% compared to BMD and composite reference standard, respectively. For SensiTest™, the EA’s were 93% and 90%. For UMIC, 87% and 90%, and for Micronaut, 83% and 84%. All four tests demonstrated categorical agreement (CA) above 90%. CA for SensiTest™ and Micronaut was both 96%, UMIC 94%, and Sensititre™ 93%. All tests were reproducible as tested in two quality control isolates. In conclusion, in clinical isolates from a large referral center, the four commercial tests for determination of colistin minimum inhibitory concentrations showed acceptable performance.

Evaluation of Two Commercial Broth Microdilution Methods Using Different Interpretive Criteria for the Detection of Molecular Mechanisms of Acquired Azole and Echinocandin Resistance in Four Common Candida Species.

The abilities of the new Vitek 2 AST-YS08 (YS08) and Sensititre YeastOne (SYO) systems to detect the resistances of Candida isolates to azoles and echinocandins were evaluated. In total, 292 isolates, including 28 Candida albicans (6 Erg11 and 2 Fks mutants), 57 Candida parapsilosis (26 Erg11 mutants), 24 Candida tropicalis (10 Erg11 and 1 Fks mutants), and 183 Candida glabrata (39 Pdr1 and 13 Fks mutants) isolates, were tested. The categorical agreements (CAs) between the Clinical and Laboratory Standards Institute (CLSI) method and YS08 fluconazole MICs obtained using clinical breakpoints were 92.4% (C. albicans), 96.5% (C. parapsilosis), and 87.0% (C. tropicalis), and the CAs between the CLSI and SYO MICs were 92.3% (C. albicans), 77.2% (C. parapsilosis), 100% (C. tropicalis), and 98.9% (C. glabrata). For C. glabrata, the CAs with the CLSI micafungin MICs were 92.4% and 55.5% for the YS08 micafungin and caspofungin MICs, respectively; they were 100%, 95.6%, and 98.9% for the SYO micafungin, caspofungin, and anidulafungin MICs, respectively. YS08 does not provide fluconazole data for C. glabrata; the CA with the CLSI fluconazole MIC was 97.8% for the YS08 voriconazole MIC, using an epidemiological cutoff value (ECV) of 0.5 μg/ml. Increased CAs with the CLSI MIC were observed for the YS08 MIC using CLSI ECVs (for fluconazole and C. tropicalis, 100%; for micafungin and C. glabrata, 98.9%) and for the SYO MIC using method-specific ECVs (for fluconazole and C. parapsilosis, 91.2%; for caspofungin and C. glabrata, 98.9%). Therefore, the YS08 and SYO systems may have different abilities to detect mechanisms of azole and echinocandin resistance in four Candida species; the use of method-specific ECVs may improve the performance of both systems.

Comparison of Antimicrobial Susceptibility Testing Methods for Colistin against Carbapenem Resistant Enterobacteriaceae in a Tertiary Care Hospital of Southern India

Introduction: The emergence of resistance to colistin urges the need for standardised invitro susceptibility testing methods for colistin, both for patient care and also for epidemiological purpose. Many challenges have been faced by clinical laboratories in performing and interpreting Antimicrobial Susceptibility Testing (AST) methods for colistin. To overcome the challenges, a rapid and reliable method is required to carry out AST for colistin. Aim: To determine the degree of agreement and types of errors associated with various colistin antimicrobial susceptibility methods such as commercial Broth Microdilution (BMD) method (MIKROLATEST- Erba Mannheim colistin MIC kit) and in-house Rapid polymyxin NP test (named after P. Nordmann and L. Poirel) with the reference in-house BMD method. Materials and Methods: The study was carried out in a tertiary care hospital in southern India from July 2018 to July 2019. The isolates from Enterobacteriaceae family showing resistance to carbapenem were selected for the study. A total of 294 clinical isolates were collected and subjected to two test methods (Commercial BMD method and Rapid polymyxin NP test) and finally the comparative analytical study was done by comparing the results of the test methods with reference in-house BMD method. The statistical analysis was carried out with the help of Stata version 14 using kappa statistics. Results: Susceptibility results of 294 isolates included Escherichia coli (n=67), K.pneumoniae (n=195), Enterobacter spp. (n=23), Citrobacter spp. (n=9) were evaluated for three methods according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) published clinical breakpoints. Overall, categorical agreement of commercial BMD with reference in-house method was 91.1% with very major error 2.7% and major error 6.1% and essential agreement was 83.3%. With the Rapid polymyxin NP test, categorical agreement, sensitivity and specificity were 92.5%, 93.1% and 92.3% respectively. Conclusion: This study helps in introducing less laborious, faster and reliable susceptibility method for colistin. This will guide the clinicians in appropriate and judicious use of colistin so that mortality and morbidity will be reduced drastically.

Genotyping and antimicrobial resistance of Streptococcus uberis isolated from bovine clinical mastitis.

A genotypic characterization of Streptococcus uberis isolated from clinical mastitis (CM) in dairy cows, and the association of Strep. uberis genotypes and antimicrobial susceptibility (AMS) was performed. A total of 89 isolates identified as Strep. uberis from 86 dairy cows with CM in 17 dairy herds of Southeastern Brazil were genotyped using random amplified polymorphic DNA (RAPD) analysis. After genotyping, two clusters (I and II) were created according to RAPD types. A commercial broth microdilution test was used to determine the susceptibility of Strep. uberis isolates to 8 antimicrobials (ampicillin, ceftiofur, cephalothin, erythromycin, penicillin, penicillin+novobiocin, pirlimycin and tetracycline). For each antimicrobial, we determined the minimal inhibitory concentrations that inhibit 50% (MIC50) and 90% (MIC90) of Strep. uberis strains. Differences in AMS among genotypic clusters were evaluated using mixed regression models. Overall, a great polymorphism (56 RAPD-types) was found among Strep. uberis isolates, although a higher genetic similarity (based on the PCR bands features) was observed within herds after genotypic clustering. No differences in AMS were observed among clusters. Strep. uberis isolated from bovine CM were resistant to most antimicrobials, with the exception of cephalothin and penicillin+novobiocin.