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Introduction: 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. Materials and methods: Two commercial BMD panels for testing of multidrugresistant Gram-negative bacteria were compared: the Thermo Scientific™ Sensititre DKMGN and the Beckman Coulter NMDRM1, for 17 antimicrobial agents. Results: 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. Discussion: Overall, the categorical agreement (CA) and essential agreement (EA) were both above 90%, but several useful antibiotics for the treatment of multiresistant 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. [ABSTRACT FROM AUTHOR]

Abstract The Clinical and Laboratory Standards Institute (CLSI) M27 guidelines are the recommended and most commonly used protocols for broth microdilution antifungal susceptibility testing of yeasts. However, these guidelines are limited to the use of 96-well assay plates, limiting assay capacity. With the increased risk of fungal resistance emerging in the community, it is important to have alternative protocols available, that offer higher throughput and can screen more than eight to ten potential antifungal compounds per plate. This study presents an optimised broth microdilution minimum inhibitory concentration (MIC) method for testing the susceptibility of yeasts in an efficient high throughput screening setup, with minimal growth variability and maximum reproducibility. We extend the M27 guidelines and optimise the conditions for 384-well plates. Validation of the assay was performed with ten clinically used antifungals (fluconazole, amphotericin B, 5-fluorocytosine, posaconazole, voriconazole, ketoconazole, itraconazole, caspofungin diacetate, anidulafungin and micafungin) against Candida albicans and Cryptococcus neoformans.

Purpose: The aim of this study was to compare the broth microdilution method (BMD)-based commercial system and colistin broth disk elution method (CBDE) for colistin susceptibility in multidrug resistant Gram-negative bacteria. Materials and methods: 112 K. pneumoniae and 62 A. baumannii isolates which were isolated from various clinical specimens between March 2020 and June 2022 were included in the study. The colistin susceptibilities of the strains were compared with BMD-based commercial system and CBDE methods. Results: For K. pneumoniae isolates, a very good agreement was found between the results of the KSDE test and those of the commercially available SMD-based kit (κ=0.88). However, for A. baumannii isolates, there was a negligible level of agreement between the results of the KSDE test and the commercially available SMD-based kit (κ=0.11). Conclusions: The CBDE method is highly compatible in determining the colistin susceptibility of Klebsiella species. The low level of agreement in Acinetobacter isolates indicates that new methods that can be applied in the routine laboratory as an alternative to the gold standard BMD method, are needed.

The objectives of this study were to investigate the effects of group housing (three calves per group) on bovine respiratory disease (BRD), diarrhea and antimicrobial resistance (AMR) to fecal commensal Escherichia coli (EC) and enterococci/streptococci (ES). Our study comprised two arms, one experimental and one observational. In the experimental arm, preweaned calves on a California dairy were randomized to either individual (IND; n = 21) or group (GRP; n = 21) housing, using a modified California-style wooden hutch. The study period lasted from birth to 56 days of age, during which calves were health scored daily. Cumulative incidence and hazard ratios were estimated for disease. Antimicrobial resistance outcomes were assessed using a prospective cohort design; feces were collected from each calf three times per week and EC and ES were evaluated for AMR using the broth microdilution method against a panel of 19 antimicrobial drugs (AMD). Analysis of treatment records was used to select calves that had been exposed (EXP) to an AMD-treated calf. In GRP, exposure occurred when a calf was a hutchmate with an AMD-treated calf. In IND, exposure occurred when a calf was a neighbor with an AMD-treated calf (TRT). Age-matched unexposed calves (UNEXP) were then selected for comparison. Proportions of AMR in fecal commensals among EXP, UNEXP, and TRT calves were compared between GRP and IND. Accelerated failure time survival regression models were specified to compare differences in minimum inhibitory concentration (MIC) of fecal commensals between EXP and UNEXP calves within each of GRP and IND calves separately. Group calves had a BRD hazard 1.94 times greater that of IND calves (p = 0.03), using BRD treatment records as the outcome. For AMR in EC isolates, higher resistance to enrofloxacin was detected in enrofloxacin-EXP GRP isolates compared with enrofloxacin-EXP IND isolates, and UNEXP GRP calves had lower resistance to ceftiofur compared with enrofloxacin-EXP and enrofloxacin-TRT calves. A significant housing-by-time interaction was detected for EC ceftiofur MIC in EXP GRP calves at 4-14 days post exposure to enrofloxacin (MIC EXP-UNEXP: µg/mL (95% CI): 10.62 (1.17, 20.07)), compared with UNEXP calves. The findings of this study show an increase in BRD hazard for group-housed calves and an increase in ceftiofur resistance in group-housed calves exposed to an enrofloxacin-treated calf.

IntroductionAntimicrobial 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.Materials and methodsTwo 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.ResultsA 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.DiscussionOverall, 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.

ABSTRACT The minimum inhibitory concentration (MIC) of echinocandins against Aspergillus spp. does not represent the actual inhibition threshold of echinocandins. Therefore, the recommended method to evaluate their activity is determining the minimum effective concentration (MEC) in broth microdilution, a method that is less common in clinical settings. This study aimed to assess a user-friendly commercial method, Sensititre YeastOne (SYO), to determine the effectiveness of echinocandins (caspofungin, anidulafungin and micafungin) against Aspergillus spp. Echinocandins MEC was determined against 23 isolates of Aspergillus spp. using SYO and the reference Clinical and Laboratory Standards Institute (CLSI) method. MECs were read with an inverted microscope and a reading mirror. Essential agreement (EA) between the tested methods was defined as a ±twofold dilution difference. There was a high EA (91%–100%) between the reference method and SYO in determining echinocandins MEC against Aspergillus isolates using inverted microscopy. A high EA was also observed between SYO MEC determined by inverted microscopy and a reading mirror, but different incubation times were required. SYO is a reliable, simple method for determining the MEC of echinocandins against Aspergillus isolates, preferably with an inverted microscope, and can be easily used in clinical laboratories when echinocandin susceptibility testing is required.IMPORTANCEUsing a commercial method such as Sensititre YeastOne (SYO) to determine the minimum effective concentration (MEC) of echinocandins against Aspergillus spp. has been shown to be a reliable alternative to the Clinical and Laboratory Standards Institute (CLSI) reference method. This makes it more suitable for high-volume clinical laboratories. SYO provides accurate results comparable to the standard method and could potentially improve patient care by guiding more optimal antifungal treatment choices for patients with Aspergillus infections.

BackgroundPseudomonas aeruginosa is a common pathogen causing healthcare-associated infections. Most surveillance studies from Saudi Arabia that assessed the resistance by P. aeruginosa were conducted in single centers or did not use broth microdilution (BMD), the gold standard test. This is the first national multicenter study to assess the resistance profiles of P. aeruginosa isolates in Saudi Arabia using BMD.MethodsBetween 2022 and 2023, isolates from various infection sites were collected from seven hospitals in seven different regions of Saudi Arabia. The isolates were shipped to an academic microbiology lab, where their susceptibility was tested by BMD following Clinical Laboratory Standards Institute guidelines using Sensititre GNX3F plates. %Susceptibility to each antibiotic, and MIC50 and MIC90 were determined.ResultsIn total, 185 P. aeruginosa isolates were collected. Most isolates came from respiratory specimens (34.1%), followed by urine (21.1%) and skin/soft tissue (17.8%). The highest susceptibility was to amikacin (76.8%). Concurrently, susceptibility to meropenem was 52%, but it was 43.8% to colistin. While all P. aeruginosa isolates met the definition of multidrug-resistance, 41 (22.2%) were difficult-to-treat and 10 (5.4%) were pandrug-resistant. Difficult-to-treat isolates made up 30.3% of skin and soft tissue isolates, 25.4% of respiratory isolates, 21.7% of blood isolates, and 17.9% of urine isolates.ConclusionPseudomonas aeruginosa demonstrated an unexpectedly high level of resistance to several commonly used antibiotics. Therefore, antimicrobial stewardship and infection control policies should be strictly enforced by hospitals across the country to optimize treatment, prevent the emergence and spread of resistant strains, and track resistance trends with local antibiograms.

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ABSTRACT Despite the first-line recommendation of fosfomycin for uncomplicated urinary tract infections (UTIs), there are pressing barriers for optimizing its use for the treatment of non-Escherichia coli Enterobacterales UTI. There are no approved breakpoints for oral use against other Enterobacterales, and the recommended agar dilution (AD) reference method for minimal inhibitory concentration (MIC) determination is largely impractical. Using 160 clinical Klebsiella pneumoniae isolates, we sought to understand rates of skipped wells and MIC imprecision in broth microdilution (BMD) and how that compares to rates of error using AD. Though the Clinical and Laboratory Standards Institute refers to the skipped well phenomena in their recommendation against the use of BMD, there is a paucity of data on its frequency. While AD and BMD produced similar MIC50/90 values (32/256 µg/mL for AD and 64/256 µg/mL for BMD), essential agreement was poor. No-growth wells at concentrations below the MIC occurred in up to 10.9% of wells at a given concentration, as the most frequent scientific error. Growth in concentrations above the measured MIC occurred in up to 3.3% of wells and was seen within three dilutions of the MIC for BMD. Observation of single colonies either at or beyond the measured MIC for AD was also common and occurred up to 8.3% and 2.5% of the time, respectively. The frequent scientific error in both testing methods should prompt re-evaluation of AD guidelines and expansion of MIC testing methods for fosfomycin susceptibility testing, as poor agreement with another method prone to scientific error should not be the main detractor from BMD use.IMPORTANCEDespite the recommendation of fosfomycin for uncomplicated urinary tract infections (UTIs), there are barriers for optimizing its use. There are no approved breakpoints for oral use against other Enterobacterales, and the recommended agar dilution (AD) reference method for MIC determination is largely impractical. The use of broth microdilution (BMD) for fosfomycin testing is not recommended by the Clinical and Laboratory Standards Institute due to unsatisfactory precision and skipped wells—occurrence of no-growth in a single well before the minimal inhibitory concentration (MIC)—and trailing endpoints. We sought to understand rates of skipped wells and growth at concentrations above measured MICs in BMD and how that compares to scientific error using AD. No-growth wells at concentrations below the MIC occurred in up to 10.9% of wells for BMD and single colonies at or beyond measured MICs for AD were also common. Frequent scientific error in both methods should prompt re-evaluation of both AD and BMD for fosfomycin susceptibility testing.

Objective: In the report published by the Clinical Laboratory Standards Institute-European Committee on Antimicrobial Susceptibility Testing (EUCAST), Polymyxin Breakpoints Working Group, they recommended that laboratories using semi-automatic devices take into account the manufacturer's recommendations and implement strict quality control (QC) studies when reporting the colistin result. In this study, we compared the one-year colistin susceptibility results with those of Sensititre FRCOL and Phoenix M50. Thus, we aimed to determine the reliability of Phoenix M50 for reporting colistin results. Material and Methods: Extensively drug-resistant Gram-negative bacteria grown from clinical samples that arrived at the laboratory between June 2021 and June 2022 were included. MALDI- TOF Microflex LT/ SH Smart MS was used for bacterial identification, and Phoenix M50 and Sensititre FRCOL were used for colistin antibiotic susceptibility testing, according to the manufacturer's recommendations. The results obtained were evaluated in line with the EUCAST criteria. QC was performed using Escherichia coli ATCC 25922 and NCTC 13846 strains in accordance with EUCAST recommendations. Results: We studied 782 strains of K. pneumoniae (n=175), P. aeruginosa (n=99), and A. baumannii (n=508). Categorical agreements were 90.3%, 93.9%, and 94.5%. The very major error rate (VME) of Phoenix M50 was found to be 40.4%. Considering the VME for K. pneumoniae, A. baumannii, and P. aeruginosa, 17.7%, 75.0%, and 100.0% were found, respectively. The ME rates of K. pneumoniae, A. baumannii, and P. aeruginosa were 5.3%, 0.8%, and 1.1%, respectively. Conclusion: The susceptible colistin results of these bacteria by Phoenix M50 should be confirmed by broth microdilution as the VME is above acceptable values. While the results of colistin detection resistant by Phoenix M50 could be reported for P. aeruginosa and A. baumannii, it needs to be confirmed with broth microdilution for K. pneumoniae.

Burkholderia pseudomallei is intrinsically resistant to many antibiotics. This study aimed to assess bacterial colony morphotypes and the validity of using disk diffusion method (DD) to determine antibiotic resistance in Malaysian clinical B. pseudomallei isolates for ceftazidime (CAZ), meropenem (MEM), amoxicillin-clavulanate (AMC) and doxycycline (DOX). DD produced good categorical agreements exhibiting concordance of 100% with reference method, broth microdilution for CAZ and DOX, 98.6% for MEM and 97.2% for AMC. Smooth-centred colonies were most frequently observed. EUCAST DD interpretative criterion is suitable to interpret B. pseudomallei CAZ, MEM, AMC and DOX resistance. Increasing AMC MIC in B. pseudomallei is a concern. [ABSTRACT FROM AUTHOR]

This research investigated the antibacterial activities of AgFe2O4 and Fe3O4 ferrite nanoparticles compared to AgFe2O4/SiO2/Passiflora Caerulea and Fe3O4/SiO2/Passiflora Caerulea nanocomposites. To synthesize ferrite nanocomposites, Passiflora Caerulea plant extract was doped onto ferrite nanoparticles with the assistance of silica (SiO2). The degree of crystallinity, phase composition, microstructure, and the compositions of the samples were determined using X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, field emission scanning electron microscopy (FESEM), and energy dispersive X-ray analysis (EDXA), respectively. Furthermore, the broth microdilution method was employed against Gram-positive and Gram-negative bacteria to assess the antimicrobial activity. The method was also applied to Gram-positive and Gram-negative bacteria to examine the antimicrobial activity. The results of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of silver and iron nanocomposites indicated that these nanocomposites exhibited superior antibacterial activity compared to silver and iron ferrite nanoparticles. Thus, silver and iron ferrite nanocomposites could serve as a novel antibacterial agent against infectious bacteria. [ABSTRACT FROM AUTHOR]

Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future. [ABSTRACT FROM AUTHOR]

Polymyxin E is the main drug used in the treatment of microorganisms with multidrug resistance. The positively charged nature of colistin creates difficulties in susceptibility testing. As a result of the studies, the gold standard method to determine colistin susceptibility was determined as the broth microdilution method. However, this method has not yet entered the laboratory routine in many centers in our country. Our aim in this review was to examine methods that could detect colistin susceptibility, which was very important in treatment, in the light of current data. Disk diffusion method is not recommended due to the limited diffusion of the colistin molecule into the agar. While there are studies suggesting that E-test can be a reliable and suitable alternative when compared to the reference method, there are also studies that detect a very major error rate of over 3%. Automated methods have not been able to achieve the desired categorical agreement and very major error rates in many studies. Molecular-based methods are especially used to detect the mcr gene and are especially important in determining resistance transmission. The findings of newly developed rapid tests and methods such as colistin broth disk elution are encouraging, but more studies are needed in this area.

Background. Among invasive fungal infection pathogens, Candida spp. represent the most common aetiological agents. The increasing rate of severe infections and the emergence of antimicrobial resistance highlight the importance of in vitro susceptibility testing. The EUCAST and the CLSI have established reference microdilutions that are reliable but difficult to apply in a laboratory routine. Commercial microdilutions could represent a valuable alternative within a diagnostic workflow. Methods. A number of 50 Candida spp. collected from positive blood samples simultaneously underwent the Sensititre Yeast-One microdilution as a standard susceptibility test and the Micronaut-AM as an experimental method. A comparison between the two techniques was produced, evaluating the effectiveness of the Micronaut-AM compared to the extensively consolidated Sensititre Yeast-One. Results. The two techniques revealed optimal agreement rates, confirming the reliability of the commercial microdilution kits within the diagnostic workflows. The results showed remarkable concordance for both susceptible and resistant isolates, highlighting slight variations in the different identified Candida species. Conclusions. Future studies about antifungal susceptibility testing should be encouraged, including molecular confirmation of possible resistance phenotypes and extended isolate numbers for the different Candida species. Moreover, it would be interesting to plan clinical trials after the execution of the examined commercial microdilution methods.

ABSTRACT The aim of this study was to evaluate the correlation between inhibitory zones and minimum inhibitory concentrations (MICs) when testing cefiderocol against Acinetobacter baumannii complex using disk diffusion and the broth microdilution (BMD) method according to the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Four-hundred and sixty-eight non-duplicated A. baumannii complex clinical isolates were collected from 56 hospitals of the China Antimicrobial Surveillance Network from 2019 to 2021. BMD using iron-depleted cation-adjusted Mueller-Hinton broth (ID-CAMHB) and standard disk diffusion methods were performed according to CLSI guidelines. Results were interpreted according to the CLSI and EUCAST breakpoints. Categorical agreement (CA), minor error (mE), major error (ME), and very major error (VME) were calculated for disk diffusion methods. The susceptibilities of all A. baumannii complex isolates by BMD were 98.7% (CLSI) and 97.6% (EUCAST). For all A. baumannii complex isolates, CA was 98.1% (CLSI) and 97.0% (EUCAST), with 0.9% (CLSI) and 1.9% (EUCAST) of VME, respectively. For the carbapenem-susceptible A. baumannii complex, the CA was 100%, with no mE or VME using both CLSI and EUCAST breakpoints. For carbapenem-resistant A. baumannii complex, CA was 97.5% (CLSI) and 96.2% (EUCAST), with 1.1% (CLSI) and 2.5% (EUCAST) of VME, respectively. Regarding the difficult-to-treat resistance A. baumannii complex isolates, CA was 97.6% (CLSI) and 95.7% (EUCAST), with 1.2% (CLSI) and 3.1% (EUCAST) of VME, respectively. Cefiderocol disk diffusion was difficult to assess in this study. Very few isolates were resistant to cefiderocol by BMD using CLSI breakpoint, and these were categorized as susceptible with the disk diffusion test. This study did, however, show that the main proportion of A. baumannii isolates were susceptible to cefiderocol by BMD, including carbapenem-resistant A. baumannii. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii is a major global health concern due to its high prevalence and limited treatment options. Cefiderocol is the only novel Food and Drug Administration (FDA)-approved β-lactam agent for the salvage treatment of carbapenem-resistant A. baumannii infection. Currently, a commercial automated susceptibility testing panel of cefiderocol is unavailable. Both the preparation of iron-depleted cation-adjusted Mueller-Hinton broth and the performance of broth microdilution are cumbersome in routine microbiology laboratories. A disk diffusion method is convenient for cefiderocol antimicrobial susceptibility testing, but limited data are available specifically for A. baumannii clinical isolates. Moreover, the Clinical and Laboratory Standards Institute published revisions to the A. baumannii cefiderocol disk diffusion breakpoints in 2022. Hence, we evaluated the performance of cefiderocol disk diffusion compared with the reference BMD against A. baumannii clinical isolates, especially those with cefiderocol zone diameters ≤ 14 mm.

Background: over the past decade, the dependency on colistin as a last resort antibiotic has driven the global emergence of colistin resistance among many bacterial species. This study comparatively evaluated the colistin Minimum Inhibitory Concentration (MIC) by various phenotypic methods, including the reference method of Broth Microdilution (BMD), other approved methods of Colistin Broth Disk Elution (CBDE), and Colistin Agar Test (CAT) and widely available method of Epsilometer Test (E-test) among Multidrug Resistant (MDR) Gram-negative bacteria. Methods: ninety Gram-negative bacterial isolates that were resistant to three or more classes of drugs (MDR) were included in the study. All the MDR isolates were subjected to colistin susceptibility determination by BMD, CBDE, CAT, and E-test. Results: amongst 1118 samples, 90 (8.05%) samples yielded MDR Gram-negative bacilli. All the MDR Gram-negative isolates were colistin intermediate by all four methods of phenotypic colistin susceptibility. Three Acinetobacter baumannii and two Klebsiella pneumoniae isolates that had MIC of 2 μg/mL by BMD, displayed MIC of

Objective: In the report published by the Clinical Laboratory Standards Institute-European Committee on Antimicrobial Susceptibility Testing (EUCAST), Polymyxin Breakpoints Working Group, they recommended that laboratories using semi-automatic devices take into account the manufacturer's recommendations and implement strict quality control (QC) studies when reporting the colistin result. In this study, we compared the one-year colistin susceptibility results with those of Sensititre FRCOL and Phoenix M50. Thus, we aimed to determine the reliability of Phoenix M50 for reporting colistin results. Material and Methods: Extensively drug-resistant Gram-negative bacteria grown from clinical samples that arrived at the laboratory between June 2021 and June 2022 were included. MALDI-TOF Microflex LT/SH Smart MS was used for bacterial identification, and Phoenix M50 and Sensititre FRCOL were used for colistin antibiotic susceptibility testing, according to the manufacturer's recommendations. The results obtained were evaluated in line with the EUCAST criteria. QC was performed using Escherichia coli ATCC 25922 and NCTC 13846 strains in accordance with EUCAST recommendations. Results: We studied 782 strains of K. pneumoniae (n=175), P. aeruginosa (n=99), and A. baumannii (n=508). Categorical agreements were 90.3%, 93.9%, and 94.5%. The very major error rate (VME) of Phoenix M50 was found to be 40.4%. Considering the VME for K. pneumoniae, A. baumannii, and P. aeruginosa, 17.7%, 75.0%, and 100.0% were found, respectively. The ME rates of K. pneumoniae, A. baumannii, and P. aeruginosa were 5.3%, 0.8%, and 1.1%, respectively. Conclusion: The susceptible colistin results of these bacteria by Phoenix M50 should be confirmed by broth microdilution as the VME is above acceptable values. While the results of colistin detection resistant by Phoenix M50 could be reported for P. aeruginosa and A. baumannii, it needs to be confirmed with broth microdilution for K. pneumoniae. [ABSTRACT FROM AUTHOR]

Introduction: Legionnaires' Disease is a pneumonia caused by Legionella spp., currently treated empirically with fluoroquinolones and macrolides. In this study, we aim to describe the antibiotic susceptibility pattern of environmental Legionella recovered in the south of Portugal. Methods: Minimal inhibitory concentration (MIC) determination of 57 Legionella isolates (10 Lp sg 1, 32, Lp sg 2-14 15 L. spp) was achieved by broth microdilution, as described by EUCAST, for azithromycin, clarithromycin, ciprofloxacin, levofloxacin, and doxycycline. Results: Fluoroquinolones were the most active antibiotic, displaying the lowest MIC values in contrast to doxycycline which had the highest. MIC90 and epidemiological cut-off (ECOFF) values were, respectively, 0.5/1 mg/L for azithromycin, 0.125/0.25 mg/L for clarithromycin, 0.064/0.125 mg/L for ciprofloxacin, 0.125/0.125 mg/L for levofloxacin and 16/32 mg/L for doxycycline. Discussion: MIC distributions were higher than reported by EUCAST for all antibiotics. Interestingly, two phenotypically resistant isolates with high-level quinolone resistance were identified. This is the first time that MIC distributions, lpeAB and tet56 genes have been investigated in Portuguese environmental isolates of Legionella. [ABSTRACT FROM AUTHOR]

Background. Among invasive fungal infection pathogens, Candida spp. represent the most common aetiological agents. The increasing rate of severe infections and the emergence of antimicrobial resistance highlight the importance of in vitro susceptibility testing. The EUCAST and the CLSI have established reference microdilutions that are reliable but difficult to apply in a laboratory routine. Commercial microdilutions could represent a valuable alternative within a diagnostic workflow. Methods. A number of 50 Candida spp. collected from positive blood samples simultaneously underwent the Sensititre Yeast-One microdilution as a standard susceptibility test and the Micronaut-AM as an experimental method. A comparison between the two techniques was produced, evaluating the effectiveness of the Micronaut-AM compared to the extensively consolidated Sensititre Yeast-One. Results. The two techniques revealed optimal agreement rates, confirming the reliability of the commercial microdilution kits within the diagnostic workflows. The results showed remarkable concordance for both susceptible and resistant isolates, highlighting slight variations in the different identified Candida species. Conclusions. Future studies about antifungal susceptibility testing should be encouraged, including molecular confirmation of possible resistance phenotypes and extended isolate numbers for the different Candida species. Moreover, it would be interesting to plan clinical trials after the execution of the examined commercial microdilution methods. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) has become a topic of great concern in recent years, with much effort being committed to developing alternative treatments for resistant bacterial pathogens. Drug combinational therapies have been a major area of research for several years, with modern iterations using combining well-established antibiotics and other antimicrobials with the aim of discovering complementary mechanisms. Previously, we characterised four GRAS antimicrobials that can withstand thermal polymer extrusion processes for novel medical device-based and therapeutic applications. In the present study, four antimicrobial bioactive—silver nitrate, nisin, chitosan and zinc oxide—were assessed for their potential combined use as an alternative synergistic treatment for AMR bacteria via a broth microdilution assay based on a checkerboard format. The bioactives were tested in arrangements of two-, three- and four-drug combinations, and their interactions were determined and expressed in terms of a synergy score. Results have revealed interesting interactions based on treatments against recognised test bacterial strains that cause human and animal infections, namely E. coli, S. aureus and S. epidermidis. Silver nitrate was seen to greatly enhance the efficacy of its paired treatment. Combinations with nisin, which is a lantibiotic, exhibited the most interesting results, as nisin has no effect against Gram-negative bacteria when used alone; however, it demonstrated antimicrobial effects when combined with silver nitrate or chitosan. This study constitutes the first study to both report on practical three- and four-drug combinational assays and utilise these methods for the assessment of established and emerging antimicrobials. The novel methods and results presented in this study show the potential to explore previously unknown drug combination compatibility measures in an ease-of-use- and high-throughput-based format, which can greatly help future research that aims to identify appropriate alternative treatments for AMR, including the screening of potential new bioactives biorefined from various sources. [ABSTRACT FROM AUTHOR]

Currently, there is an increasing number of cases of fungal infections caused by opportunistic strains of the yeast Rhodotorula mucilaginosa, mainly in immunocompromised patients during hospitalization. The excessive use of antibiotics and azole compounds increases the risk of resistance to microorganisms. A new alternative to these drugs may be synthetic phthalide lactones with a structure identical to or similar to the natural ones found in celery plants, which show low toxicity and relatively high fungistatic activity. In the present study, the fungistatic activity of seven phthalide lactones was determined against R. mucilaginosa IHEM 18459. We showed that 3-n-butylidenephthalide, the most potent compound selected in the microdilution test, caused a dose-dependent decrease in dry yeast biomass. Phthalide accumulated in yeast cells and contributed to an increase in reactive oxygen species content. The synergistic effect of fluconazole resulted in a reduction in the azole concentration required for yeast inhibition. We observed changes in the color of the yeast cultures; thus, we conducted experiments to prove that the carotenoid profile was altered. The addition of lactones also triggered a decline in fatty acid methyl esters. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is a global threat to healthcare, and despite the availability of new drugs, polymyxins are still an important therapeutic option for this and other resistant gram-negative pathogens. Broth microdilution is the only method that is recommended for polymyxins. In this study, we evaluated the accuracy of a commercial Policimbac® plate in determining the polymyxin B MIC for K. pneumoniae clinical isolates. The results were compared with those of the broth microdilution method according to ISO 16782. The Policimbac® plate had an excellent 98.04% categorical agreement, but unacceptable 31.37% essential agreement rates. Almost 2% of major errors as observed. Additionally, 52.94% of the strains overestimated the MIC at 1 µg/mL. Three isolates were excluded from the analysis due to the drying of the Policimbac® plate. To avoid dryness, we included wet gauze for the test, obtaining a 100% of categorical agreement rate; however, a low essential agreement was maintained (25.49%). In conclusion, the Policimbac® plate was unable to correctly determine the polymyxin B MIC for K. pneumoniae isolates. This low performance may interfere with the clinical use of the drug and, thus, with the result of the patient's treatment. [ABSTRACT FROM AUTHOR]

For 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. We 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. The 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. As 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. [ABSTRACT FROM AUTHOR]

Qi Zhang,1,* Wenjuan Yan,1,* Yingjie Zhu,2,* Nan Jing,1 Shanmei Wang,1 Youhua Yuan,1 Bing Ma,1 Junhong Xu,1 Yafei Chu,1 Jiangfeng Zhang,1 Qiong Ma,1 Baoya Wang,1 Wenbo Xu,1 Liqiang Zhu,3 Ying Sun,2 Caiqin Shi,4 Juan Fang,5 Yi Li,1 Shengqun Liu6 1Department of Clinical Microbiology, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, and People’s Hospital of Henan University, Zhengzhou, People’s Republic of China; 2Department of Clinical Microbiology, Henan No.3 Provincial People’s Hospital, Zhengzhou, People’s Republic of China; 3Department of Clinical Microbiology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China; 4Department of Microbiology Laboratory, KingMed Diagnostics Group Co., Ltd, Zhengzhou, People’s Republic of China; 5Department of Research and Development, Autobio Diagnostics Co., Ltd, Zhengzhou, People’s Republic of China; 6Department of Anesthesia and Perioperative Medicine, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, and People’s Hospital of Henan University, Zhengzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yi Li; Shengqun Liu, Department of Anesthesia and Perioperative Medicine, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, and People’s Hospital of Henan University, Weiwu Road 7, Zhengzhou, Henan, 450003, People’s Republic of China, Tel +8613526882118, Fax +8637187160318, Email liyilabmed@henu.edu.cn; tim2002627@163.comPurpose: To evaluate the performance of five widespread commercial products for colistin and polymyxin B susceptibility testing in China for mcr-positive and -negative Escherichia coli and Klebsiella pneumoniae.Methods: A total of 132 E. coli and 83 K. pneumoniae strains (including 68 mcr-1-positive E. coli and 28 mcr-8-positive K. pneumoniae) were collected. We analysed the performance of colistin susceptibility (with Vitek 2 and Phoenix M50) and the performance of polymyxin B susceptibility (with DL-96II, MA120, and a Polymyxin B Susceptibility Test strip; POL E-strip). Broth microdilution was used as the gold standard. Categorical agreement (CA), essential agreement (EA), major error (ME), and very major error (VME) were calculated for comparisons.Results: For E. coli, the total CA, EA, ME, and VME to colistin were as follows: Vitek 2, 98.5%/98.5%/0%/2.9%; and Phoenix M50, 98.5%/97.7%/0%/2.9%. The total CA, EA, ME, and VME to polymyxin B were as follows: POL E-strip, 99.2%/63.6%/1.6%/0%; MA120, 70.0%/-/0%/58.8%; and DL-96II, 80.2%/-/1.6%/36.8%. Only Vitek 2 and Phoenix M50 presented satisfactory performances for mcr-1-positive E. coli. For K. pneumoniae, the total CA, EA, ME, and VME to colistin were as follows: Vitek 2, 73.2%/72.0%/0%/61.6%; and Phoenix M50, 74.7%/74.7%/0%/58.3%. The total CA, EA, ME, and VME to polymyxin B were as follows: POL E-strip, 91.6%/74.7%/2.1%/16.7%; MA120, 92.8%/-/2.1%/13.9%; and DL-96II, 92.2%/-/2.1%/8.3%. All systems were unsatisfactory for mcr-8-positive K. pneumoniae. When the susceptibility of mcr-negative strains was tested, all systems presented excellent performance.Conclusion: Vitek 2 and Phoenix M50 with colistin for E. coli showed acceptable performance regardless of mcr-1 expression, while DL-96II, MA120, and the POL E-strip performed worse for mcr-1-positive strains. Furthermore, mcr-8 greatly affected the performance of all systems with both colistin and polymyxin B for K. pneumoniae isolates.Keywords: polymyxin B, colistin, mobilised colistin resistance, broth microdilution, susceptibility testing, semi-automated systems

Gayatree Nayak, Bijayini Behera, Srujana Mohanty, Punyatoya Kar, Jayanti Jena Department of Microbiology, All India Institute of Medical Sciences [AIIMS], Bhubaneswar, Odisha, 751019, IndiaCorrespondence: Bijayini Behera, Department of Microbiology, All India Institute of Medical Sciences [AIIMS], Bhubaneswar, Odisha, 751019, India, Tel +07750847562, Email drbinny2004@gmail.comIntroduction: Cefiderocol (CFDC), a novel semi-synthetic siderophore cephalosporin has been developed to combat the menace of infections caused by carbapenem-resistant Gram-negative bacilli (CR-GNB) including Carbapenem-resistant Enterobacterales (CRE) and Carbapenem-resistant Nonfermenting Gram-negative bacilli (CR-NFGNB).Methods: We determined the in vitro activity of CFDC against a contemporary collection of 503 CR-GNB isolates by the reference broth microdilution method (BMD) using Iron depleted cation adjusted Mueller-Hinton broth (ID-CAMHB). Performance of CFDC disk diffusion (DD) was evaluated against the reference BMD, as an alternative convenient testing method. Molecular characterization of carbapenemase in CR-GNB was performed by PCR targeting blaNDM-1, blaOXA-48like alleles, blaKPC, blaIMP, and, blaVIM. Minimum inhibitory concentration (MIC) distribution of CFDC in CR-GNB harbouring different carbapenemase enzymes was also analyzed.Results: In our study, 81.7% (411/503) of CR-GNB isolates [81.3%, (278/342) CRE and 82.6% (133/161) CR-NFGNB] were susceptible to CFDC (p> 0.05). Categorical agreement (CA) of DD ranged from 79.8% to 87.5%, Minor error (mE) ranged from 0 to 14%, Major error (ME) ranged from 0 to 3.5%, and Very Major error (VME) ranged from 0 to 12.5% with variations by species tested. Overall CFDC MIC50 and MIC90 values of CR-GNB isolates without any carbapenemase genes were higher as compared to those with the presence of carbapenemase genes (4 μg/mL and 128 μg/mL versus 2 μg/mL and 16 μg/mL respectively).Discussion: CFDC is not yet available for clinical use in India. Hence, multicentric studies are the need of the hour in India for standardization of CFDC susceptibility using disks and CAMHB from different manufacturers as well as understanding mechanisms of high MIC values.Keywords: Cefiderocol, carbapenem-resistant Gram negative bacilli, broth microdilution, carbapenemases, disk diffusion, minimum inhibitory concentration

Background: over the past decade, the dependency on colistin as a last resort antibiotic has driven the global emergence of colistin resistance among many bacterial species. This study comparatively evaluated the colistin Minimum Inhibitory Concentration (MIC) by various phenotypic methods, including the reference method of Broth Microdilution (BMD), other approved methods of Colistin Broth Disk Elution (CBDE), and Colistin Agar Test (CAT) and widely available method of Epsilometer Test (E-test) among Multidrug Resistant (MDR) Gram-negative bacteria. Methods: ninety Gram-negative bacterial isolates that were resistant to three or more classes of drugs (MDR) were included in the study. All the MDR isolates were subjected to colistin susceptibility determination by BMD, CBDE, CAT, and E-test. Results: amongst 1118 samples, 90 (8.05%) samples yielded MDR Gram-negative bacilli. All the MDR Gram-negative isolates were colistin intermediate by all four methods of phenotypic colistin susceptibility. Three Acinetobacter baumannii and two Klebsiella pneumoniae isolates that had MIC of 2 µg/mL by BMD, displayed MIC of <1 µg/mL by CBDE and CAT. Three isolates (2 Citrobacter koseri and 1 Enterobacter spp.) showed higher MIC by the E-strip method in comparison to BMD. Conclusions: our study holds significance, as there is a paucity of data comparing the four phenotypic methods for colistin MIC determination; BMD is the most reliable, gold standard method, but it is labor-intensive and requires technical expertise. In the present study, CBDE and CAT methods showed good concordance with BMD, and are easy to perform with limited logistics. Thus, they can be used as an alternative to BMD. We found that even though the Etest method was less accurate, it can still be used with caution to exclude the possibility of colistin resistance. [ABSTRACT FROM AUTHOR]

Antimicrobial susceptibility testing (AST) is a critical function of the clinical microbiology laboratory and is essential for optimizing care of patients with infectious diseases, monitoring antimicrobial resistance (AMR) trends, and informing public health initiatives. Several methods are available for performing AST including broth microdilution, agar dilution, and disk diffusion. Technological advances such as the development of commercial automated susceptibility testing platforms and the advent of rapid diagnostic tests have improved the rapidity, robustness, and clinical application of AST. Numerous accrediting and regulatory agencies are involved in the process of AST and setting and revising breakpoints, including the U.S. Food and Drug Administration and the Clinical and Laboratory Standards Institute. Challenges to optimizing AST include the emergence of new resistance mechanisms, the development of new antimicrobial agents, and generation of new data requiring updates and revisions to established methods and breakpoints. Together, the challenges in AST methods and their interpretation create important opportunities for well‐informed clinicians to improve patient outcomes and provide value to antimicrobial stewardship programs, especially in the setting of rapidly changing and increasing AMR. Addressing AST challenges will involve continued development of new technologies along with collaboration between clinicians and the laboratory to facilitate optimal antimicrobial use, combat the increasing burden of AMR, and inform the development of novel antimicrobials. This updated primer serves to reinforce important principles of AST, and to provide guidance on their implementation and optimization. [ABSTRACT FROM AUTHOR]

Conducting antimicrobial susceptibility testing (AST) in a comparable manner requires the availability of a standardized method. Organizations, such as the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), provide standardized protocols for a range of fastidious bacteria but not for Mycoplasma hyorhinis. We developed a broth microdilution method for testing M. hyorhinis in a standardized and harmonized way using a modified Friis broth devoid of antimicrobial or otherwise bacterial growth-inhibiting agents. The type strain M. hyorhinis DSM 25591 was chosen to establish the methodology. The antimicrobial agents of interest were doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin, tested by using commercial SensititreTM microtiter plates. In addition, the suitability of the methodology was evaluated via variation of the individual ingredients of the modified Friis broth by either using different batches or choosing other distributors. Despite these alterations, the method provided reliable results. We obtained repeatable minimal inhibitory concentrations for all six tested field isolates and the M. hyorhinis type strain. With this newly proposed method, we aim to provide an improved AST method for diagnostic laboratories and monitoring purposes with better comparability between times and countries. In addition, this new method will allow for an improvement of targeted treatments using antimicrobial agents and thereby reduce the options for resistance development. [ABSTRACT FROM AUTHOR]

Introduction: Colistin is increasingly used as a last-choice antimicrobial in the treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa isolates. Due to the recent increase in colistin resistance around the world, the need for reliable methods for susceptibility testing has become more important. In this study, it was aimed to determine the performance of the broth disc elution (BDE) test, which was developed as an alternative to the reference broth microdilution (BMD) method. Materials and Methods: Fourty eight multidrug resistant Pseudomonas aeruginosa strains isolated from various clinical specimens were included in the study. The BMD method was performed according to the recommendations of the ISO-standard (20776-1), and the BDE test was done according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. The categorical agreement, major error, and very major error (VME) rates of the BDE test were determined by comparing with the BMD method. Results: By using the reference BMD method, 47 of 48 isolates were found to be susceptible (98%), and one was found to be resistant (2%). According to BMD test, the minimum inhibitory concentration distribution of the isolates was between 0.25-64 mg/l. The BDE test also correctly detected 47 susceptible and 1 resistant isolates. When the BDE test was compared with the BMD, the rate of categorical agreement was found to be 100%. No major or VMEs were detected. Conclusion: The BDE test performed very well in determining colistin susceptibility in multidrug resistant P. aeruginosa isolates. Therefore, we think that the BDE test can be used as a practical and reliable method in laboratories with limited resources where it is not possible to perform the reference BMD method.

ABSTRACT Chlorhexidine bathing to prevent transmission of multidrug-resistant organisms has been adopted by many U.S. hospitals, but increasing chlorhexidine use has raised concerns about possible emergence of resistance. We sought to establish a broth microdilution method for determining chlorhexidine MICs and then used the method to evaluate chlorhexidine MICs for bacteria that can cause health care-associated infections. We adapted a broth microdilution method for determining chlorhexidine MICs, poured panels, established quality control ranges, and tested Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae complex isolates collected at three U.S. sites. Chlorhexidine MICs were determined for 535 isolates including 129 S. aureus, 156 E. coli, 142 K. pneumoniae, and 108 E. cloacae complex isolates. The respective MIC distributions for each species ranged from 1 to 8 mg/L (MIC50 = 2 mg/L and MIC90 = 4 mg/L), 1 to 64 mg/L (MIC50 = 2 mg/L and MIC90 = 4 mg/L), 4 to 64 mg/L (MIC50 = 16 mg/L and MIC90 = 32 mg/L), and 1 to >64 mg/L (MIC50 = 16 mg/L and MIC90 = 64 mg/L). We successfully adapted a broth microdilution procedure that several laboratories were able to use to determine the chlorhexidine MICs of bacterial isolates. This method could be used to investigate whether chlorhexidine MICs are increasing. IMPORTANCE Chlorhexidine bathing to prevent transmission of multidrug-resistant organisms and reduce health care-associated infections has been adopted by many hospitals. There is concern about the possible unintended consequences of using this agent widely. One possible unintended consequence is decreased susceptibility to chlorhexidine, but there are not readily available methods to perform this evaluation. We developed a method for chlorhexidine MIC testing that can be used to evaluate for possible unintended consequences.

Abstract Background Tuberculosis (TB) is a communicable, preventable and curable disease caused by the bacterium Mycobacterium tuberculosis (MTB). Peru is amongst the 30 countries with the highest burden of multidrug-resistant tuberculosis (MDR-TB) worldwide. In the fight against drug-resistant tuberculosis, the UKMYC6 microdilution plate was developed and validated by the CRyPTIC project. The objective of the study was to evaluate the use of the broth microdilution (BMD) plate methodology for susceptibility testing of drug-resistant MTB strains in Peru. Methods MTB strains isolated between 2015 and 2018 in Peru were used. 496 nationally-representative strains determined as drug-resistant by the routine 7H10 Agar Proportion Method (APM) were included in the present study. The Minimum Inhibitory Concentration (MIC) of 13 antituberculosis drugs were determined for each strain using the UKMYC6 microdilution plates. Diagnostic agreement between APM and BMD plate methodology was determined for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. Phenotypes were set using binary (or ternary) classification based on Epidemiological cut-off values (ECOFF/ECV) proposed by the CRyPTIC project. Whole Genome Sequencing (WGS) was performed on strains with discrepant results between both methods. Results MIC distributions were determined for 13 first- and second-line anti-TB drugs, including new (bedaquiline, delamanid) and repurposed (clofazimine, linezolid) agents. MIC results were available for 80% (397/496) of the strains at 14 days and the remainder at 21 days. The comparative analysis determined a good agreement (0.64 ≤ k ≤ 0.79) for the drugs rifampicin, ethambutol, ethionamide and kanamycin, and the best agreement (k > 0.8) for isoniazid and levofloxacin. Overall, 12% of MIC values were above the UKMYC6 plate dilution ranges, most notably for the drugs rifampicin and rifabutin. No strain presented MICs higher than the ECOFF/ECV values for the new or repurposed drugs. Discrepant analysis using genotypic susceptibility testing by WGS supported half of the results obtained by APM (52%, 93/179) and half of those obtained by BMD plate methodology (48%, 86/179). Conclusions The BMD methodology using the UKMYC6 plate allows the complete susceptibility characterization, through the determination of MICs, of drug-resistant MTB strains in Peru. This methodology shows good diagnostic performances for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. It also allows for the characterization of MICs for other drugs used in previous years against tuberculosis, as well as for new and repurposed drugs recently introduced worldwide.

Klebsiella pneumoniae is a global threat to healthcare, and despite the availability of new drugs, polymyxins are still an important therapeutic option for this and other resistant gram-negative pathogens. Broth microdilution is the only method that is recommended for polymyxins. In this study, we evaluated the accuracy of a commercial Policimbac® plate in determining the polymyxin B MIC for K. pneumoniae clinical isolates. The results were compared with those of the broth microdilution method according to ISO 16782. The Policimbac® plate had an excellent 98.04% categorical agreement, but unacceptable 31.37% essential agreement rates. Almost 2% of major errors as observed. Additionally, 52.94% of the strains overestimated the MIC at 1 µg/mL. Three isolates were excluded from the analysis due to the drying of the Policimbac® plate. To avoid dryness, we included wet gauze for the test, obtaining a 100% of categorical agreement rate; however, a low essential agreement was maintained (25.49%). In conclusion, the Policimbac® plate was unable to correctly determine the polymyxin B MIC for K. pneumoniae isolates. This low performance may interfere with the clinical use of the drug and, thus, with the result of the patient’s treatment.

IntroductionLegionnaires’ Disease is a pneumonia caused by Legionella spp., currently treated empirically with fluoroquinolones and macrolides. In this study, we aim to describe the antibiotic susceptibility pattern of environmental Legionella recovered in the south of Portugal.MethodsMinimal inhibitory concentration (MIC) determination of 57 Legionella isolates (10 Lp sg 1, 32, Lp sg 2-14 15 L. spp) was achieved by broth microdilution, as described by EUCAST, for azithromycin, clarithromycin, ciprofloxacin, levofloxacin, and doxycycline. ResultsFluoroquinolones were the most active antibiotic, displaying the lowest MIC values in contrast to doxycycline which had the highest. MIC90 and epidemiological cut-off (ECOFF) values were, respectively, 0.5/1 mg/L for azithromycin, 0.125/0.25 mg/L for clarithromycin, 0.064/0.125 mg/L for ciprofloxacin, 0.125/0.125 mg/L for levofloxacin and 16/32 mg/L for doxycycline.DiscussionMIC distributions were higher than reported by EUCAST for all antibiotics. Interestingly, two phenotypically resistant isolates with high-level quinolone resistance were identified. This is the first time that MIC distributions, lpeAB and tet56 genes have been investigated in Portuguese environmental isolates of Legionella.

The pygidial glands of carabids produce strong-smelling vapours. In this study, we examined the chemical composition of the gland secretions and the structure of the glands in five species of Carabini ground beetles (one species from the subtribe Calosomatina and four species from the subtribe Carabina): Calosoma (Calosoma) maderae (Fabricius, 1775), Carabus (Carabus) granulatus Linnaeus, 1758, C. (Limnocarabus) clathratus Linnaeus, 1760, C. (Carabus) ulrichii Germar, 1823, and C. (Procerus) gigas Creutzer, 1799. Additionally, we tested the antibacterial potential of the pygidial gland secretions of the two latter species against 11 bacterial strains. In order to detect the chemical content of the secretions, we used gas chromatography–mass spectrometry (GC–MS). The secretion extracts were applied against selected strains of medically important bacteria. We used bright-field microscopy to examine the morphology of the glands. We discovered a total of 11 chemical compounds in the pygidial gland extracts of the ground beetles we analysed. Ten of these compounds were identified as seven carboxylic acids, two hydrocarbons, and one aromatic aldehyde, while one chemical remained unidentified. Most of the components were isolated from the secretion of C. (L.) clathratus (nine), while the lowest number of compounds was found in C. (P.) gigas (two). Methacrylic acid was the most dominant compound by percentage in all five species, while angelic acid was also detected in all samples. As expected, salicylaldehyde was exclusively found in the species of the genus Calosoma Weber, 1801. The secretion of C. (P.) gigas was shown to achieve the highest level of antibacterial activity against Pseudomonas aeruginosa, Salmonella enterica, and S. typhimurium (even the same level as the positive control streptomycin), while the secretion of C. (C.) ulrichii achieved the highest antimicrobial potential against Staphylococcus epidermidis, S. aureus, Listeria monocytogenes, and Bacillus cereus. The most noticeable difference in the structure of the glands between the two genera is that the reservoir in Calosoma is more significantly narrowed as it leads into the efferent duct, compared to that of Carabus.

Leptospira interrogans is a biofilm-forming pathogen, however, there are few data involving Brazilian strains isolated from dogs and their antimicrobial sensitivity in planktonic and biofilm forms. The potential for biofilm formation and antimicrobial resistance in naturally infected dogs is a fundamental approach towards disease epidemiology and the establishment of consistent prophylaxis and control measures. The objective of this study was to evaluate in vitro biofilm formation of a reference strain (L. interrogans, sv. Copenhageni L1 130 - L20) and of L. interrogans isolated from dogs (C20, C29, C51, C82), with subsequent evaluation of antimicrobial susceptibility in planktonic and biofilm forms. The semi quantification of biofilm production revealed a dynamic process of development over time, with mature biofilm formation early on the seventh day of incubation. All strains were efficient for in vitro biofilm formation and, in this form, they were considerably more resistant compared to their planktonic form, with MIC90 of 1600 μg/mL for amoxicillin, 800 μg/mL for ampicillin, and >1600 μg/mL for doxycycline and ciprofloxacin. The strains studies were isolated on naturally infected dogs that might act as reservoirs and sentinels for human infections. The potential to antimicrobial resistance together with the close relation between dogs and humans indicates the need for greater actions on disease control and surveillance. Moreover, biofilm formation may contribute to the persistence of Leptospira interrogans in the host and these animals can act as chronic carriers, disseminating the agent in the environment.

Delay in the results of standard phenotypic susceptibility tests is the main obstacle to adequate antibiotic treatment. For this reason, the European Committee for Antimicrobial Susceptibility Testing has proposed the Rapid Antimicrobial Susceptibility Testing for the disk diffusion method directly from blood culture. However, to date, there are no studies evaluating early readings of polymyxin B broth microdilution (BMD), the only standardized methodology for assessing susceptibility to polymyxins. This study aimed to evaluate modifications in the BMD technique for polymyxin B using fewer antibiotic dilutions and reading after an incubation time of 8–9 hr (early reading) in comparison to 16–20 hr of incubation (standard reading) for isolates of Enterobacterales, Acinetobacter baumannii complex, and Pseudomonas aeruginosa. A total of 192 isolates of gram-negative bacteria were evaluated and the minimum inhibitory concentrations were read after early and standard incubations. The early reading presented 93.2% of essential agreement and 97.9% of categorical agreement with the standard reading of BMD. Only three isolates (2.2%) presented major errors and only one (1.7%) presented a very major error. These results indicate a high agreement between the early and the standard reading times of BMD of polymyxin B. [ABSTRACT FROM AUTHOR]

The emergence of antimicrobial resistance (AMR) is increasingly common across the globe and aquatic ecosystems could be considered a reservoir of antibiotic-resistant bacteria. This study aimed to determine prevalence and antibiotic susceptibility of the potential pathogenic bacteria Salmonella spp. and Vibrio spp. in bivalve molluscs intended for human consumption, collected over a period of 19 months along the northern coast of Apulia region. The AMR profile was also determined in non-pathogenic Vibrio species, common natural inhabitants of seawater and a useful indicator for the surveillance of AMR in the environment. The current study presents data on the AMR of 5 Salmonella and 126 Vibrio isolates by broth microdilution MIC. Multidrug resistance (MDR) was observed in one S. Typhimurium strain towards sulfamethoxazole, trimethoprim, tetracycline, gentamicin, and ampicillin and in 41.3% of the Vibrio strains, mostly towards sulphonamides, penicillin, and cephems. All Vibrio isolates were sensitive to azithromycin, chloramphenicol, tetracycline, amoxicillin/clavulanic acid, gentamicin, streptomycin, amikacin, and levofloxacin. The AMR phenomenon in the investigated area is not highly worrying but not entirely negligible; therefore, in-depth continuous monitoring is suggested. Results concerning the antibiotic agents without available specific clinical breakpoints could be useful to upgrade the MIC distribution for Vibrio spp. but, also, the establishment of interpretative criteria for environmental species is necessary to obtain a more complete view of this issue. [ABSTRACT FROM AUTHOR]

Background: The reference broth microdilution (rBMD) method for the determination of colistin resistance is very laborious and time consuming, and many manual errors can occur. There are also limitations in detection of colistin heteroresistance. Therefore, alternative methods with satisfactory performance are required for routine laboratory work. In our study, the colistin broth disk elution (CBDE) method recommended by the Clinical and Laboratory Standards Institute (CLSI) for the detection of colistin resistance in routine applications was compared with rBMD. The compatibility and error rates of the method were evaluated and its usability in routine laboratory studies was examined. Methods: Eighty-nine multidrug resistant Klebsiella pneumoniae and five Echerichia coli strains isolated from various clinical specimens were included in the study. Identification of strains and antibiotic susceptibility tests were performed with MALDI-TOF MS (bioMerieux, France) and Vitek-2 (bioMerieux) system. Minimum inhibitory concentration (MIC) was studied in 0.125 - 128 mg/L dilution range by using polystyrene microplate and colistin sulfate salt according to ISO-standard (20776-1) recommendations for the reference BMD test. The CBDE method was performed according to the CLSI recommendations. Isolates with MIC ≤ 2 mg/L were considered susceptible, while isolates with MIC > 2 mg/L were considered resistant according to EUCAST recommendations. The performance of the CBDE method was evaluated according to ISO criteria (Categorical agreement > 90%; major error and very major error rates < 3%). Results: Categorical agreement for all 58 and 36 isolates found to be resistant and susceptible, respectively, by rBMD was found to be 100% with CBDE test. Since < 1 and > 4 µg/mL values could not be determined with the CBDE method, essential agreement (EA) could not be calculated. No major or very major errors were detected. Conclusions: Our results showed that the performance of the CBDE test is good when compared to the rBMD method. According to our data, we believe that the CBDE method can be used in routine laboratories for the detection of colistin resistance. [ABSTRACT FROM AUTHOR]

Background Helicobacter pylori infection is an infectious disease and thus the eradication treatment should be guided by susceptibility testing. This study aimed to assess the applicability of broth microdilution as a routine susceptibility testing method for H. pylori. Methods Susceptibility profiles of clarithromycin (CLR) and levofloxacin (LEV) resistance in 76 clinical H. pylori isolates were simultaneously assessed using agar dilution and broth microdilution methods. The correlation between the minimum inhibitory concentrations (MICs) obtained by the 2 methods was assessed by means of linear regression analysis. Results The correlation between the MICs determined by broth microdilution method and agar dilution method was good for both CLR (r = 0.966) and LEV (r = 0.959). The susceptibility agreement between the 2 methods was 100% for CLR and 96.1% for LEV. Using the broth microdilution method, the false resistance was found in 3.9% (3 of 76) strains for LEV susceptibility testing. No false susceptibility was found for either CLR or LEV, and no false resistance was found for susceptibility testing of CLR. Conclusions The broth microdilution method is suitable for routine susceptibility testing of clinical H. pylori isolates. [ABSTRACT FROM AUTHOR]

Azithromycin is an alternative to treat invasive non-typhoidal Salmonella (iNTS) infections. We determined its epidemiological cut-off (ECOFF) and compared azithromycin susceptibility testing methods for iNTS. We used EUCAST ECOFFinder to determine the minimum inhibitory concentrations (MIC; obtained by broth microdilution) ECOFF and corresponding disk zone diameters of 515 iNTS from blood cultures in Democratic Republic of Congo, Burkina Faso, Rwanda, and Cambodia. Transferable resistance mechanisms were determined by polymerase chain reaction. We compared azithromycin susceptibility testing by semi-automated broth microdilution (customized Sensititre panel; reference), agar dilution, gradient tests (bioMérieux, Liofilchem, HiMedia; read at 80% (MIC80%) and 100% inhibition (MIC100%)), and disk diffusion (Rosco, Oxoid, BD, Liofilchem) for 161 wild- and 198 non–wild-type iNTS. Azithromycin MIC ECOFF was 16 mg/L corresponding to a 12 mm zone diameter; mphA was detected in 192/197 non–wild- and 0/47 wild-type iNTS. Categorical agreement was excellent (≥98%) for all methods. Essential agreement was very good for agar dilution (>90%) but moderate for gradient tests (MIC80%: 52% to 71% and MIC100%: 72% to 91%). Repeatability was good for all methods/brands. Interreader agreement was high for broth microdilution and agar dilution (all ≤1 twofold dilution difference) and disk diffusion (>96% ≤3 mm difference) but lower for gradient tests (MIC80% & MIC100%: 83% to 94% ≤1 twofold dilution difference). Azithromycin ECOFF of iNTS was 16 mg/L, i.e. equal to Salmonella Typhi. Disk diffusion is an accurate, precise, and user-friendly alternative for agar dilution and broth microdilution. Reading gradient tests at 100% instead of 80% inhibition improved accuracy and precision. [ABSTRACT FROM AUTHOR]

Yersiniosis is an important zoonotic disease; however, data are scarce on the resistance of enteropathogenic yersiniae, especially that of Y. pseudotuberculosis. Minimum inhibitory concentrations (MIC) of 21 antibiotics and 3 essential oils (EOs) were determined by broth microdilution for Y. enterocolitica bioserotype 4/O:3 strains isolated from domestic swine (n = 132) and Y. pseudotuberculosis strains isolated from wild boars (n = 46). For 15 of 21 antibiotics, statistically significant differences were found between MIC values of Y. enterocolitica and Y. pseudotuberculosis. While Y. enterocolitica was more resistant to amoxiclav, ampicillin, cefotaxime, cefuroxime, gentamicin, imipenem, meropenem, tetracycline, tobramycin, and trimethoprim, Y. pseudotuberculosis was more resistant to cefepime, ceftazidime, colistin, erythromycin, and nitrofurantoin. Statistically significant differences were found between various essential oils (p < 0.001) and species (p < 0.001). The lowest MICs for multiresistant Y. enterocolitica (n = 12) and Y. pseudotuberculosis (n = 12) were obtained for cinnamon (median 414 and 207 μg/mL, respectively) and oregano EOs (median 379 and 284 μg/mL), whereas thyme EO showed significantly higher MIC values (median 738 and 553 μg/mL; p < 0.001). There was no difference between Y. enterocolitica strains of plant (1A) and animal (4/O:3) origin (p = 0.855). The results show that Y. enterocolitica is generally more resistant to antimicrobials than Y. pseudotuberculosis. [ABSTRACT FROM AUTHOR]

Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future.

Introduction: Candida auris has turned up as a multidrug-resistant nosocomial agent with outbreaks reported worldwide. The present study was conducted to evaluate the antifungal drug susceptibility pattern of C. auris. Methods: Isolates of C. auris were obtained from clinically suspected cases of candidemia from January 2019 to June 2021. Identification was done with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and panfungal DNA polymerase chain reaction (PCR), followed by sequencing. Antifungal susceptibility testing was performed with broth microdilution method. Results: Out of 50 isolates C. auris, 49 were identified by MALDI-TOF and one isolate was identified with panfungal DNA PCR followed by sequencing. For fluconazole, 84% (n = 42) isolates were found to be resistant and 16% (n = 8) isolates were susceptible (minimum inhibitory concentrations [MICs] range 0.5–16). Posaconazole exhibited potent activity, followed by itraconazole. For amphotericin B, only 6% (n = 3) isolates were resistant with MICs ≥2 μg/mL. Only 4% (n = 2) isolates exhibited resistance to caspofungin. No resistance was noted for micafungin and anidulafungin. One (2%) isolate was found to be panazole resistant. One (2%) isolate was resistant to fluconazole, amphotericin B, and caspofungin. Conclusion: Correct identification of C. auris can be obtained with the use of MALDI-TOF and sequencing methods. A small percentage of fluconazole-sensitive isolates are present. Although elevated MICs for amphotericin B and echinocandins are not generally observed, the possibility of resistance with the irrational use of these antifungal drugs cannot be denied. Pan azole-resistant and pan drug-resistant strains of C. auris are on rise.

Abstract Background Nocardia species are ubiquitous in natural environments and can cause nocardiosis. In the present study, the use of Resazurin salt and Spectrophotometry were proposed as alternative methods to reduce subjectivity in the interpretation of susceptibility results to antimicrobials by the broth microdilution method for Nocardia spp. Results The susceptibility of Nocardia spp. isolates to Amikacin, Ciprofloxacin, Minocycline and Trimethoprim-Sulfamethoxazole was evaluated by Minimum Inhibitory Concentration (MIC) determinations by the broth microdilution method. To verify cellular growth, the colour-changing dye Resazurin was applied, the Optical Densities were measured on a spectrophotometer, and both were compared to Clinical and Laboratory Standards Institute (CLSI) Gold Standard method (visual MIC determination). Percentages of essential and categorical agreements and interpretative categorical errors were calculated within each method (intra-reading) and between them (inter-reading). The Gold Standard visual reading demonstrated 100% of essential and categorical intra-reading agreements for Amikacin, and there was no error when compared with the alternative methods. For Ciprofloxacin, the comparison between the Gold Standard and the Spectrophotometric reading showed 91.5% of essential agreement. In the categorical intra-reading analysis for Minocycline, there were 88.1 and 91.7% in the Gold Standard and in the Spectrophotometric readings, respectively, and 86.4% of concordance between them. High rates of categorical agreement were also observed on the Trimethoprim-Sulfamethoxazole analyses, with 93.7% for the Gold Standard, 84.9% for the Resazurin readings, and 80.5% between them. Conclusions The alternative methods with Resazurin and Spectrophotometric readings showed high agreement rates with the Gold Standard.