CLSI Reference Method Research Articles

In vitro activity of sulopenem and comparator agents against Enterobacterales and anaerobic clinical isolates collected during the SENTRY Antimicrobial Aurveillance Program.

Physicians must leverage several factors when making antibiotic therapy decisions, including route of administration and duration of therapy. Oral administration provides several potential advantages including increased accessibility, prevention of hospitalizations and earlier discharges. Sulopenem-a broad-spectrum, synthetic penem β-lactam agent-uniquely possesses both oral and IV formulations along with noted stability among antimicrobial-resistant subsets. This study evaluated the in vitro activity of sulopenem and comparator agents against contemporary Enterobacterales and anaerobic clinical isolates predominantly from patients with bloodstream, intra-abdominal and urinary tract infections. A contemporary collection of 1647 Enterobacterales and 559 anaerobic isolates was assembled from medical centres in Europe and the USA. Isolates were susceptibility tested using the CLSI reference methods: broth microdilution for Enterobacterales and agar dilution for anaerobes. Sulopenem demonstrated potent in vitro antimicrobial activity (MIC50/90, 0.03/0.25 mg/L) against Enterobacterales isolates regardless of infection type, inhibiting 99.2% of isolates at ≤1 mg/L. This activity was conserved against resistant phenotypes including ESBL-phenotype Escherichia coli (MIC50/90, 0.03/0.06 mg/L) and ESBL-phenotype Klebsiella pneumoniae (MIC50/90, 0.06/1 mg/L). Sulopenem maintained activity against ciprofloxacin-, nitrofurantoin- and trimethoprim/sulfamethoxazole-non-susceptible subsets (MIC50/90, 0.03-0.06/0.12-0.5 mg/L). Against anaerobic isolates, sulopenem (98.9% inhibited at ≤4 mg/L) and meropenem [98.4% susceptible (CLSI)] were the most active compounds tested. The potent in vitro activity of sulopenem against this large collection of recent Enterobacterales and anaerobic clinical isolates from multiple infection types supports its further clinical evaluation in the treatment of intra-abdominal and urinary tract infections.

1672. Antimicrobial Activity of Cefepime-Taniborbactam and Comparators Against Clinical Isolates from ICU and Non-ICU Patients; 2018-2020 Global Surveillance

Abstract Background Taniborbactam is a novel cyclic boronate-based broad-spectrum β-lactamase inhibitor with selective direct inhibitory activity against both serine- and metallo-β-lactamases. Taniborbactam restores the activity of cefepime against many difficult to treat organisms, including cephalosporin- and carbapenem-resistant Enterobacterales (EB) and P. aeruginosa (PA). The activity of cefepime-taniborbactam (FTB) and comparator agents was evaluated against clinical isolates of EB and PA from a 2018-2020 global surveillance study. Methods 16,742 isolates of EB and PA were collected from patients in wards designated ICU and non-ICU in 2018-2020. MICs of FTB and comparators were determined by the CLSI reference method. Resistant (R) phenotypes were based on 2022 CLSI breakpoints. Multidrug resistant (MDR) was defined as resistance to at least one agent from ≥3 drug classes. Provisional susceptible (S) breakpoints of ≤8 µg/mL and ≤16 µg/mL were applied for FTB for comparative purposes only. Results FTB had potent activity against EB, with MIC90s of 0.25 µg/mL for isolates from non-ICU patients and 0.5 µg/mL for isolates from ICU patients (Table). At least 99.2% of all EB from both wards were inhibited by ≤8 µg/mL FTB. FTB maintained activity against R phenotypes of EB in both ward types, with 90.3% to 98.1% of isolates inhibited at ≤8 µg/mL. FTB had potent activity against PA, with MIC90 values of 8 µg/mL and inhibiting ≥94.0% of isolates at ≤8 µg/mL for both ICU and non-ICU isolates. FTB maintained activity against R phenotypes of PA in both ward types, with 71.0% to 83.7% of isolates inhibited at ≤8 µg/mL and 84.7% to 92.5% inhibited at ≤16 µg/mL. %S to comparator agents against the R subsets ranged from 1.6% to 92.6% for EB, and 36.0% to 68.4% for PA. Against the particularly challenging subset of MDR PA from ICU patients, FTB was the most active agent tested as it inhibited 73.5/84.7% of isolates at ≤8/≤16 µg/mL compared to 43.1%S to CZA and 36.0%S to C/T. Conclusion Cefepime-taniborbactam demonstrated potent in vitro activity against EB and PA from ICU and non-ICU wards, including resistant subsets of isolates. This supports the continued development of FTB as a potential new treatment option for infections due to resistant Gram-negative pathogens. Disclosures All Authors: No reported disclosures.

1693. Meropenem-Xeruborbactam: In Vitro Potency against Gram-Negative Bacteria in Comparison with Marketed and Investigational Beta-lactam (BL)/Beta-lactamase Inhibitor (BLI) Combinations

Abstract Background Several BLI combinations have entered into clinical testing or been approved for use but there are few comparative studies of their in vitro activity. Xeruborbactam (XER, formerly QPX7728) is a cyclic boronate BLI with potent inhibitory activity against serine and metallo-beta-lactamases (MBL). It is in clinical development in combination with meropenem (MEM). The objective of this study was to compare in vitro potency of MEM-XER to that of other investigational and approved BL/BLI combinations in head-to-head testing against recent isolates of carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii (CRAB) as well as Pseudomonas aeruginosa (PA). Methods The test panel of recent worldwide isolates consisted of 507 CRE including 168 MBL producers (157 NDM, 11 VIM, 1 IMP), 506 CRAB, and 506 PA that were representative of current isolates and included 65 MDR (resistant to ≥ 3 antimicrobial classes) strains. MEM-XER was tested with XER at a fixed 8 mg/L conc. and other BL/BLI combinations at their recommended BLI concentrations. MIC testing was conducted blinded to drug identity using CLSI reference methods and controlled using approved quality control bacterial strains and ranges. Results The MIC50 and MIC90 for MEM-XER vs CRE ranged from ≤ 0.03 mg/L to 0.06 mg/L and 0.06 mg/L to 1 mg/L, respectively. MEM-XER and cefepime-taniborbactam (FEP-TANI) were the only BL/BLIs with activity against MBL-producers; based on MIC90, MEM-XER (MIC90 of 1 mg/L) was 16-fold more potent than FEP-TANI (MIC90 of 16 mg/L). MEM-XER and sulbactam-durlobactam (SUL-DUR) were the only agents with high potency against CRAB, with MIC50/MIC90 of 0.5/4 mg/L and 1/2 mg/L. For the representative panel of PA, the potency of MEM-XER, FEP-TAN, ceftolozane-tazobactam (TOL-TAZ), imipenem-relebactam (IMP-REL), and CAZ-AVI was similar with MIC90s ranging between 2-8 mg/L; for the MDR panel, MIC90‘s were > 8 mg/L for all combinations, with MER-XER, IMP-REL, TOL-TAZ, and CAZ-AVI having MIC50’s of 2-4 mg/L. Conclusion Compared to the marketed or investigational BL/BLIs, MEM-XERU had the broadest coverage of carbapenem-resistant gram-negative bacteria, with the most differentiating property being activity against multiple strains of MBL-producing CRE and CRAB. Disclosures Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Jill Lindley, BS, AbbVie: Grant/Research Support.

353. Evaluation of the in vitro activity of amphotericin B used in the liposomal formulation AmBisome against contemporary human fungal pathogens

Abstract Background Amphotericin B is an antifungal drug whose main limitation is toxicity. To reduce these side effects, liposomal formulations have been developed, being AmBisome (L-AmB, Gilead Science Inc) the most commonly used. We describe the in vitro antifungal activity of L-AmB component used in the AmBisome formulation Methods We performed a multicenter study to determine the susceptibility to L-AmB using CLSI and EUCAST reference methods. Contemporary human fungal pathogenic species (14 yeast spp and 23 mould spp from different geographical regions were included. Other antifungals (triazoles and echinocandins) were also evaluated. Results L-AmB showed activity against yeast species using both EUCAST and CLSI protocols. The strongest activity was shown against Candida albicans (GM-EUCAST 0.24 mg/L (n=85), CLSI 0.23 mg/L (n=111)), C. parapsilosis (GM-EUCAST 0.32 mg/L (n=82), CLSI 0.32 mg/L (n=124)), C. glabrata (GM-EUCAST 0.32 mg/L (n=84), CLSI 0.38 mg/L (n=145)), C. auris (GM-EUCAST 0.46 mg/L (n=20), CLSI 0.31 mg/L (n=22)). In filamentous fungi, lowest MICs were found for Aspergillus fumigatus (GM-EUCAST 0.62 mg/L (n=77), CLSI 0.65 mg/L (n=124)), and A. niger (GM-EUCAST 0.25 mg/L (n=72) , CLSI 0.32 mg/L (n=85)), while for other Aspergillus species, there was a higher variability in the MICs (A. flavus, GM-EUCAST 1.2 mg/L (n=71), CLSI 1.75 mg/L (n=76); A. terreus GM-EUCAST 1.7 mg/L (n=65), CLSI 1.04 mg/L (n=71); A. nidulans GM-EUCAST 1.38 mg/L (n=39), CLSI 0.73 mg/L (n=39)). For other genera from filamentous fungi, L-AmB had a strong activity (GM around 0.2-0.5 mg/L for both methods, including Talaromyces marneffei, Rhizopus arrhizus and R. microscoporus, Lichtheimia corymbifera, Mucor circinelloides). The less susceptible species corresponded to multi-resistant (MDR) species, such as Lomentospora prolificans and Cunninghamella bertholletiae. Conclusion The L-AmB component of AmBisome showed activity against most contemporary human fungal pathogens and only showed limited activity against some MDR mould species. Disclosures Oscar Zaragoza, Principal Investigator, Gilead: Grant/Research Support Teresa Merino-Amador, n/a, Gilead: Grant/Research Support Cristina de Armentia, n/a, Gilead: Grant/Research Support Cornelia Lass-Flörl, PhD, Gilead: Grant/Research Support Jochem B. Buil, PhD, Gilead: Grant/Research Support Paul E. Verweij, PhD, Gilead: Grant/Research Support Patrick C. Y. Woo, PhD, Gilead: Grant/Research Support Chi-Ching Tsang, PhD, Gilead: Grant/Research Support P. Lewis White, PhD, Associates of Cape Cod: Honoraria|F2G: Advisor/Consultant|Gilead: Grant/Research Support|IMMY: Honoraria|Pfizer: Advisor/Consultant|Pfizer: Honoraria Jessica Price, PhD, Gilead: Grant/Research Support Carolyn Grimes, PhD, Gilead: Grant/Research Support Alessandro C. Pasqualotto, PhD, Gilead: Grant/Research Support Manuel Cuenca-Estrella, PhD, Gilead: Grant/Research Support.

Real world clinical feasibility of direct-from-specimen antimicrobial susceptibility testing of clinical specimens with unknown microbial load or susceptibility

Within healthcare settings, physicians use antibiograms, which offer information on local susceptibility rates, as an aid in selecting empirical antibiotic therapy and avoiding the prescription of potentially ineffective drugs. While antibiograms display susceptibility and resistance data at hospital, city, or region-specific levels and ultimately enable the initiation of antibiogram-based empirical antibiotic treatment, AST reports at the individual patient level and guides treatments away from broad-spectrum antibiotics towards narrower-spectrum antibiotics or the removal of antibiotics entirely. Despite these advantages, AST traditionally requires a 48- to 72-h turn-around; this window of time can be critical for some antimicrobial therapeutic interventions. Herein, we present a direct-from-specimen AST to reduce the time between patient sampling and receipt of lab AST results. The biggest challenge of performing AST directly from unprocessed clinical specimens with an unknown microbial load is aligning the categorical susceptibility report with CLSI reference methods, which start from a fixed inoculum of 0.5 McFarland units prepared using colonies from a sub-culture. In this pilot clinical feasibility study using de-identified remnant specimens collected from MCW, we observed the high and low ends of microbial loads, demonstrating a final categorical agreement of 87.5% for ampicillin, 100% for ciprofloxacin, and 100% for sulfamethoxazole-trimethoprim.

719. Susceptibility Testing of Oteseconazole (VT-1161) Against Clinical Isolates from Phase 3 Clinical Studies in Subjects with Recurrent Vulvovaginal Candidiasis

Abstract Background Oteseconazole (VT-1161) is a novel, investigational oral therapy that is currently under FDA review for the treatment of recurrent vulvovaginal candidiasis (RVVC). Susceptibility testing was performed on all viable clinical isolates collected from three Phase 3 studies to determine the susceptibility of causative pathogens to oteseconazole versus the current treatment standard of care, fluconazole. Methods Vaginal cultures were obtained at screening and at all subsequent study visits throughout the duration of the studies (approx. 48 Weeks) and submitted to a central mycology laboratory for fungal species identification and storage. Susceptibility testing was conducted in accordance with the CLSI Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast M27-Ed4. Results Candida albicans was identified as the primary causative pathogen in 87% of women with RVVC presenting with an acute infection, followed by C. glabrata (8%). Other non-albicans species including; C. parapsilosis, C. tropicalis, C. krusei, C. dubliniensis, C. kefyr and Saccharomyces cerevisiae were responsible for < 1% of infections. A total of 1910 isolates were collected. The MIC range, MIC50 and MIC90 values against all isolates for oteseconazole were ≤ 0.0005 to > 0.25, 0.002 and 0.06 µg/mL, respectively. In comparison, the MIC range, MIC50 and MIC90 values for fluconazole were, ≤ 0.06 to > 32, 0.25 and 8 µg/mL respectively. The MIC range, MIC50 and MIC90 values against all C.glabrata isolates for oteseconazole were 0.002 to > 0.25, 0.03 and 0.125 µg/mL, respectively. In comparison, the MIC range, MIC50 and MIC90 values for fluconazole were, ≤ 0.06 to 32, 2 and 8 µg/mL respectively. Conclusion Oteseconazole demonstrated very low MIC values against most Candida strains, including fluconazole resistant isolates, aligning with clinical study outcomes. Oteseconazole MICs against C. glabrata strains were approximately 6-fold lower than fluconazole. Disclosures Mahmoud Ghannoum, PhD, Mycovia Pharmaceuticals (Grant/Research Support, Research Grant or Support) Thorsten Degenhardt, Ph.D, Mycovia Pharmaceuticals (Employee, Shareholder) Karen Person, M.S., Mycovia Pharmaceuticals, Inc. (Employee)Mycovia Pharmaceuticals, Inc. (Employee) Stephen Brand, Ph.D, Mycovia Pharmaceuticals (Employee)

In vitro activity of olorofim against clinical isolates of Scedosporium species and Lomentospora prolificans using EUCAST and CLSI methodologies.

To evaluate the in vitro activity of olorofim, a new broad-spectrum antifungal with a novel mechanism of action, against a collection of 123 Spanish clinical isolates belonging to five Scedosporium species and Lomentospora prolificans. The activity of olorofim against Scedosporium apiospermum (n = 30), Scedosporium boydii (n = 30), Scedosporium ellipsoideum (n = 10), Scedosporium aurantiacum (n = 20), Scedosporium dehoogii (n = 3) and Lomentospora prolificans (n = 30) was compared with that of amphotericin B, voriconazole, isavuconazole and micafungin by performing EUCAST and CLSI reference methods for antifungal susceptibility testing. Amphotericin B and isavuconazole showed MICs ≥2 mg/L for all the species evaluated and voriconazole was moderately active (GM, MIC50 and MIC90 values ≤2 mg/L) against all of them except L. prolificans. Micafungin was effective against S. apiospermum complex strains, but exhibited elevated MECs for S. dehoogii and S. aurantiacum. Olorofim showed low MICs for all the Scedosporium strains tested (GM values were lower than 0.130 and 0.339 by the EUCAST method and the CLSI method, respectively, for all of the species), including those belonging to the MDR species L. prolificans, for which GM values were 0.115 and 0.225 mg/L by the EUCAST method and the CLSI method, respectively, while the GMs for the rest of the antifungals evaluated were higher than 3.732 mg/L using both methodologies. Olorofim displayed promising in vitro activity against the Scedosporium and L. prolificans strains tested, some of which have reduced susceptibility to the antifungals that are currently in use.

Prevalence and Clonal Distribution of Azole-Resistant Candida parapsilosis Isolates Causing Bloodstream Infections in a Large Italian Hospital.

The most prevalent cause of nosocomial bloodstream infection (BSI) among non-C. albicans Candida species, Candida parapsilosis, may not only be resistant to azole antifungal agents but also disseminate to vulnerable patients. In this survey of BSIs occurring at a large Italian hospital between May 2014 and May 2019, C. parapsilosis accounted for 28.5% (241/844) of all Candida isolates causing BSI episodes. The majority of episodes (151/844) occurred in medical wards. Across the 5 yearly periods, the rates of azole non-susceptibility were 11.8% (4/34), 17.8% (8/45), 28.6% (12/42), 32.8% (19/58), and 17.7% (11/62), respectively, using the Sensititre YeastOne® method. Among azole non-susceptible isolates (54/241; 22.4%), 49 were available for further investigation. Using the CLSI reference method, all 49 isolates were resistant to fluconazole and, except one (susceptible dose-dependent), to voriconazole. Forty (81.6%) isolates harbored the Erg11p Y132F substitution and nine (18.4%) isolates the Y132F in combination with the Erg11p R398I substitution. According to their genotypes, as defined using a microsatellite analysis based on six short tandem repeat markers, 87.7% of isolates (43/49) grouped in two major clusters (II and III), whereas 4.1% of isolates (2/49) belonged to a separate cluster (I). Interestingly, all the isolates from cluster II harbored the Y132F substitution, and those from cluster III harbored both Y132F and R398I substitutions. Of 56 non-Italian isolates included as controls, two Indian isolates with the Y132F substitution had a genotype clearly differing from that of the isolates from clusters II and I. In conclusion, these findings show the dominance of clonal Y132F isolates in our hospital and suggest detection of the Y132F substitution as helpful tool to prevent transmission among hospitalized patients at risk of BSI.

2072. Multicenter Evaluation of the Etest vs. Agar Dilution for Susceptibility Testing of Helicobacter pylori

Background Helicobacter pylori is associated with peptic ulcer disease and gastric malignancy. Antimicrobial susceptibility testing (AST) is often requested for patients who fail eradication therapy. The CLSI reference method, agar dilution (AD), is not performed in most laboratories and maintaining organism viability during transit to a reference laboratory is difficult. We assessed the performance of the Etest (bioMerieux) as a method for H. pylori AST in comparison to AD.MethodsFrozen stocks of 82 H. pylori isolates with AD results previously reported by Mayo Clinic were prepared from the same plate for distribution to participating laboratories. Etest was performed at ARUP Laboratories and Cleveland Clinic (CC). For Etest, isolates were incubated for 72 hours in a microaerobic atmosphere. Aged Mueller–Hinton agar with 5% sheep blood plates were inoculated with a three McFarland suspension prepared in brain heart infusion broth. Etest strips were applied and MICs read after 72 hours of microaerobic incubation. Results were interpreted by applying CLSI and EUCAST breakpoints. Categorical agreement (CA), very major, major and minor errors (VME, ME, and mE) were determined for Etest using AD as the reference method. Isolates with errors were repeat tested in duplicate by Etest to determine the final results summarized below.ResultsFor clarithromycin, 65% of isolates were resistant (R) by AD; Etest results at each laboratory showed 97.5% CA (1 mE and 1 ME). For tetracycline, only 2.5% of isolates were R by AD; a single VME occurred at both ARUP and CC (98.8% CA) with the same isolate. The AD dilutions tested for amoxicillin prevented interpretation with EUCAST breakpoints. With one exception, amoxicillin Etest results were susceptible (S, ≤0.125 mg/L) at both laboratories (98.8% of MICs ± one dilution). Applying levofloxacin EUCAST breakpoints (S, ≤1 mg/L) to interpret ciprofloxacin results, 57.8% of isolates were R by AD. ARUP CA was 97.5% (1 ME, 1 VME) and CC CA was 96.3% (1 ME, 2 VMEs).ConclusionClarithromycin, tetracycline, and ciprofloxacin Etest results for H. pylori showed acceptable CA (>95%) at both testing sites compared with the AD reference method. The comparative ease of performance and reproducibility of the Etest may help standardize it as an AST method for H. pylori.Disclosures S. S. Richter, bioMerieux: Grant Investigator, Research grant; BD Diagnostics: Grant Investigator, Research grant; Roche: Grant Investigator, Research grant; Hologic: Grant Investigator, Research grant; Diasorin: Grant Investigator, Research grant; Accelerate: Grant Investigator, Research grant; Biofire: Grant Investigator, Research grant.

1984. Ceftriaxone-Sulbactam-EDTA vs. Meropenem: Analysis of Failed Patients With Assessment of MIC Increases and Changes in Genotypic Profile in PLEA (a Phase 3, Randomized, Double-Blind Clinical Trial in Adults With Complicated Urinary Tract Infections or Acute Pyelonephritis)

BackgroundCeftriaxone–sulbactam–EDTA (CSE) is a novel combination being developed to treat serious infections caused by Gram-negative bacteria. In vitro molecular biology studies have shown that the addition of EDTA in the combination helps to prevent horizontal gene transfer during conjugation by chelating the divalent magnesium ions (Mg2+) required for the activity of DNA relaxases enzyme. An assessment of acquisition of resistant genes and a concomitant increase in MIC for patients that failed therapy in the Phase 3 clinical trial (NCT03477422) was conducted.MethodsMICs were conducted on baseline and post-treatment isolates recovered during treatment period. MICs were determined using CLSI reference methods and MIC changes from baseline were further assessed. Bacterial DNA was extracted by the alkaline lysis method. β-Lactamase (BL) genes were amplified in single PCRs using a panel of primers for detection of most β-lactamase enzymes, including extended-spectrum β-lactamases (ESBLs) (blaTEM, blaSHV, blaCTX-M), metallo-β-lactamases (MBLs) (blaVIM, blaNDM, blaIMP), carbapenemases (blaOXA, blaKPC) and class C cephalosporinases (blaAmpC).ResultsNine of 143 [2/74 (2.7%) in CSE; 7/69 (10.1%) in MR (meropenem)] patients had a microbiological failure at the TOC visit. Of these nine patients (all E. coli), a variation in the post-treatment genotypic profile was noted for four patients (44.4%) in the MR group and two of these patients also reported a ≥4-fold increase in post-treatment MIC. Both patients harbored four distinct BL genes (blaTEM + blaSHV + blaCTX-M + blaAmpC) at baseline, and had acquired two additional genes (blaOXA, blaKPC), both carbapenemases, as a result of treatment failure (after 6 days and 8 days of IV therapy respectively) with MR. In the first case, MIC increased 16-fold (1 µg/mL to 16 µg/mL for MR and 2 µg/mL to 32 µg/mL for CSE), while in the second case, MIC increased 8-fold (1 µg/mL to 8 µg/mL) for MR and 32-fold (1 µg/mL to 32 µg/mL) for CSE. No such increase in MIC or acquisition of resistant genes was noted in patients that failed therapy with CSE.ConclusionThese findings highlight the need for an effective choice of empirical therapy as failed treatments could lead to selection for resistant genes, rendering once susceptible drug non-susceptible.Disclosures M. A. Mir, Venus Medicine Research Centre: Employee, Salary. S. Chaudhary, Venus Medicine Research Centre: Employee and Shareholder, Salary. M. Chaudhary, Venus Medicine Research Centre: Board Member and Shareholder, Salary. A. Pyasi, Venus Medicine Research Centre: Employee, Salary. R. Girotra, Venus Medicine Research Centre: Employee, Salary.

2076. Expanded Antibiotic Menu Demonstration for Novel Rapid Phenotypic Antimicrobial Susceptibility Testing Platform

BackgroundFast transitions to targeted therapies for infectious disease patients are paramount for optimal patient care and antibiotic stewardship. A next-generation phenotypic antimicrobial susceptibility test (AST) system that provides rapid results for broad menus of >30 antibiotics per patient sample is required. SeLux has developed a rapid, phenotypic AST platform that utilizes standard 384-well microplates. These consumables provide sufficient wells for simultaneous testing of newly approved antibiotics and broad selections of conventional antibiotics. Here, we demonstrate the platform’s ability to produce fast, accurate results with newly approved and not-yet-approved antibiotics.MethodsThe core of SeLux’s technology is a novel assay for bacterial surface area, which enables delineation of truly resistant bacteria from organisms that filament or swell in antibiotic concentrations above the MIC. AST was performed with the SeLux platform and compared with the CLSI broth microdilution reference method. Testing of 20 representative conventional antibiotics was performed on 1,191 isolates, including 323 FDA-CDC “challenge” strains and comprising 20 species of nonfastidious bacteria. Testing of newly developed antibiotics, generous gifts from the manufacturers, was performed with ~20 to 50 isolates with representative MICs throughout the dilution series and encompassing the breakpoint region.ResultsTesting of conventional antibiotics showed essential agreements (EA) and categorical agreements (CA) ≥90% with the CLSI reference method for all combinations tested (Figures 1 and 2). The platform returned results within 6.5 hours for >98% of the isolates tested to date. The SeLux platform’s EA was ≥90% for all newly developed antibiotics tested to date (Figure 3). For newly approved antibiotics, CAs were similarly ≥90% with no very major errors (Vmj).Figure 1Figure 2Figure 3ConclusionThe SeLux platform’s compatibility with 384-well microplates should transform the rate with which newly approved antibiotics gain use. By speeding the reporting of AST results, SeLux’s platform will further enable hospitals to simultaneously improve patient care, decrease lengths-of-stay, and meet antibiotic stewardship goals.Disclosures E. Stern, SeLux Diagnostics: Board Member, Employee and Shareholder, Salary. A. Vacic, SeLux Diagnostics: Employee and Shareholder, Salary. K. Flentie, SeLux Diagnostics: Employee and Shareholder, Salary. B. Spears, SeLux Diagnostics: Employee and Shareholder, Salary. N. Purmort, SeLux Diagnostics: Employee and Shareholder, Salary. F. Giok, SeLux Diagnostics: Employee and Shareholder, Salary. K. DaPonte, SeLux Diagnostics: Employee and Shareholder, Salary. S. Scott, SeLux Diagnostics: Employee and Shareholder, Salary. D. Puff, SeLux Diagnostics: Employee and Shareholder, Salary. F. Floyd Jr., SeLux Diagnostics: Employee and Shareholder, Salary. Z. Zhang, SeLux Diagnostics: Employee and Shareholder, Salary. P. Reilly, SeLux Diagnostics: Employee, Salary. J. Liu, SeLux Diagnostics: Employee, Salary. E. Viveiros, SeLux Diagnostics: Employee, Salary. N. Phelan, SeLux Diagnostics: Employee, Salary. C. Krebill, SeLux Diagnostics: Employee, Salary. A. Flyer, SeLux Diagnostics: Consultant, Scientific Advisor and Shareholder, Consulting fee. D. Smalley, SeLux Diagnostics: Scientific Advisor and Shareholder, Consulting fee. D. C. Hooper, SeLux Diagnostics: Scientific Advisor, Consulting fee. M. J. Ferraro, SeLux Diagnostics: Scientific Advisor and Shareholder, Consulting fee.

MYCOLOGICAL PROFILE AND ANTIFUNGAL SENSITIVITY OF INFECTIVE KERATITIS IN A TERTIARY CARE HOSPITAL OF SOUTHERN ODISHA

Background: Infective keratitis is the second major cause of blindness next to cataract. Mycotic keratitis is an important ophthalmologic problem especially in developing countries including India. Fungal infection involving cornea is a fatal condition which needs early diagnosis and treatment to save the patient’s eye. Though studies on mycotic keratitis have been reported from different part of India, to the best of our knowledge this study showing antifungal susceptibility is the first to be reported from Southern Odisha. Objective: The purpose of this study was to study epidemiological characteristics, predisposing factors, fungal etiology and antifungal susceptibility of common fungal isolates in infective keratitis cases. Materials and Methods: A prospective study was conducted from November 2015 to October2017 in the Department of Microbiology and Ophthalmology M.K.C.G Medical College and Hospital .Relevant information was recorded using standard proforma of keratitis cases. Corneal scrapings were collected under strict aseptic conditions and subjected to10% KOH mount, Gram stain and culture. Identification of fungal agents were done as per standard microbiological procedures. An antifungal sensitivity test was done by microbroth dilutions as per CLSI reference method. Results: Over a period of two years 149 patients of infective keratitis were evaluated. Microbiological diagnosis of mycotic keratitis was established in 39 (26.17%) cases. Filamentous fungi were isolated more often than yeasts. The most frequently encountered filamentous fungi and yeasts were Aspergillus spp. 14(35.89%) and Candida albicans 7 (17.94%) respectively. Males were more commonly affected and were mostly in the age group of 46-60 years. Ocular trauma due to vegetative matter was the most common predisposing factor. Natamycin was the most effective antifungal against filamentous fungi and amphotericin B was most effective for Candida albicans . Conclusion: Because of serious consequences of mycotic keratitis, it is very important to know the exact etiological agents and effective antifungals to save the eye of the patients. So laboratory confirmation should be undertaken and fungal infection should be ruled out before prescribing antimicrobial agents.

Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck.

Antifungal susceptibility testing is an essential tool for guiding therapy, although EUCAST and CLSI reference methods are often available only in specialized centers. We studied the performance of an agar-based screening method for the detection of azole resistance in Aspergillus fumigatus cultures. The VIPcheck consists of four wells containing voriconazole, itraconazole, posaconazole, or a growth control. Ninety-six A. fumigatus isolates were used. Thirty-three isolates harbored a known resistance mechanism: TR34/L98H (11 isolates), TR46/Y121F/T289A (6 isolates), TR53 (2 isolates), and 14 isolates with other cyp51A gene point mutations. Eighteen resistant isolates had no cyp51A-mediated azole resistance. Forty-five isolates had a wild-type (WT) azole phenotype. Four technicians and two inexperienced interns, blinded to the genotype/phenotype, read the plates visually after 24 h and 48 h and documented minimal growth, uninhibited growth, and no growth. The performance was compared to the EUCAST method. After 24 h of incubation, the mean sensitivity and specificity were 0.54 and 1.00, respectively, with uninhibited growth as the threshold. After 48 h of incubation, the performance mean sensitivity and specificity were 0.98 and 0.93, respectively, with minimal growth. The performance was not affected by observer experience in mycology. The interclass correlation coefficient was 0.87 after 24 h and 0.85 after 48 h. VIPcheck enabled the selection of azole-resistant A. fumigatus colonies, with a mean sensitivity and specificity of 0.98 and 0.93, respectively. Uninhibited growth on any azole-containing well after 24 h and minimal growth after 48 h were indicative of resistance. These results indicate that the VIPcheck is an easy-to-use tool for azole resistance screening and the selection of colonies that require MIC testing.

Susceptibility Testing of Common and Uncommon Aspergillus Species against Posaconazole and Other Mold-Active Antifungal Azoles Using the Sensititre Method.

We tested 59 common and 27 uncommon Aspergillus species isolates for susceptibility to the mold-active azole antifungal agents itraconazole, voriconazole, and posaconazole using the Sensititre method. The overall essential agreement with the CLSI reference method was 96.5% for itraconazole and posaconazole and was 100% for voriconazole. By the Sensititre method as well as the CLSI reference method, all of 10 A. fumigatus isolates with a cyp51 mutant genotype were classified as being non-wild-type isolates (MIC > epidemiological cutoff value [ECV]) with respect to triazole susceptibility.

Susceptibility of bacterial vaginosis (BV)-associated bacteria to secnidazole compared to metronidazole, tinidazole and clindamycin

Secnidazole, a 5-nitroimidazole with a longer half-life, is structurally related to metronidazole and tinidazole. For treatment of bacterial vaginosis (BV), secnidazole is a suitable single-dose oral drug having a longer serum half-life than metronidazole. The objective of this study was to evaluate the antimicrobial susceptibility of vaginal isolates of facultative and anaerobic bacteria to secnidazole, metronidazole, tinidazole and clindamycin.A total of 605 unique BV-related bacteria and 108 isolates of lactobacilli recovered from the human vagina of US women during the years 2009–2015 were tested for antimicrobial susceptibility by the agar dilution CLSI reference method to determine the minimal inhibitory concentration (MIC).The MIC90 (μg/mL) for secnidazole was similar to metronidazole and tinidazole for Anaerococcus tetradius (secnidazole: MIC90 2; metronidazole: MIC90 2; tinidazole: MIC90 4), Atopobium vaginae (32; >128; 128), Bacteroides species (2; 2; 2), Finegoldia magna (2; 2; 4), Gardnerella vaginalis (128; 64; 32), Mageeibacillus indolicus (2; 2; 2), Megasphaera-like bacteria (0.5; 0.25; 0.5), Mobiluncus curtisii (128; >128; >128) and Mobiluncus mulieris (>128; >128; >128), Peptoniphilus lacrimalis (4; 4; 4) and Peptoniphilus harei (2; 2; 4), Porphyromonas species (0.25; 0.5; 0.25), Prevotella bivia (8; 8; 8), Prevotella amnii (2; 1; 2) and Prevotella timonensis (2; 2; 2). In this evaluation, 14 (40%) of 35 P. bivia, 5 (14%) of 35 P. amnii and 21 (58%) of 36 P. timonensis isolates were resistant to clindamycin with MIC values of >128 μg/mL. Secnidazole, like metronidazole, was superior to clindamycin for Prevotella spp., Bacteroides spp., Peptoniphilus spp., Anaerococcus tetradius and Finegoldia magna. Clindamycin had greater activity against Atopobium vaginae, Gardnerella vaginalis and Mobiluncus spp. compared to the nitroimidazoles. All 27 Lactobacillus crispatus, 26 (96%) of 27 L. jensenii, 5 (19%) of 27 L. gasseri and 18 (67%) of 27 L. iners isolates were susceptible to clindamycin (MIC ≤2) while the MIC90 for all lactobacilli tested was >128 μg/mL for secnidazole, metronidazole and tinidazole.Secnidazole has similar in vitro activity against the range of microorganisms associated with BV compared to metronidazole or tinidazole. Further, secnidazole spares lactobacilli, a characteristic which is desirable in drugs used to treat bacterial vaginosis.

Comparative Analysis of Disc Diffusion and E-test with Broth Micro-dilution for Susceptibility Testing of Clinical Candida Isolates Against Amphotericin B, Fluconazole, Voriconazole and Caspofungin.

Antifungal susceptibility testing remains an area of intense interest because of the increasing number of clinical isolates resistant to antifungal therapy. Clinical and Laboratory Standards Institute has proposed reference broth micro dilution (BMD) method for susceptibility testing. The reference method is time-consuming and poorly suited for the routine clinical laboratory setting. Agar-based susceptibility testing methods, disk diffusion (DD) method and the E-test method can be an easier, reliable and less time consuming alternative for the BMD method. To compare the results of Amphotericin B, fluconazole, voriconazole, and Caspofungin susceptibility testing by DD, and the E-test method with the CLSI reference method for clinical Candida isolates. Broth Microdilution (BMD), E-test and Disk diffusion testing of the various clinical Candida isolates was performed in accordance with CLSI documents. The results obtained were analysed and compared. The categorical agreement for Amphotericin B, fluconazole, voriconazole, and Caspofungin susceptibility results by E-test and DD method was 65.2%, 67.4%; 100%, 82.6%; 100%, 100%; 100%, 97.8% respectively. The agar-based E-test and disk diffusion methods are reliable alternatives to the BMD method for Candida isolates when test susceptible to fluconazole, voriconazole, and Caspofungin, however the susceptibility testing results must be interpreted with caution in case of Amphotericin B.

O02.2 Susceptiblity of Bacterial Vaginosis (BV)-Associated Bacteria and Lactobacilli to Rifaximin, Metronidazole and Clindamycin

ObjectiveRifaximin is a semisynthetic rifamycin with poor oral bioavailability, used as an oral agent for the treatment of traveller’s diarrhoea and hepatic encephalopathy. Rifaximin is in development as a topical agent for the treatment of bacterial vaginosis (BV). The objective of this study was to evaluate the antimicrobial susceptibility of vaginal isolates of facultative and anaerobic bacteria to rifaximin, clindamycin and metronidazole.MethodsA total of 411 unique BV-related bacteria and 100 isolates of lactobacilli recovered from the human vagina of US women during the years 2009–2012 were tested for antimicrobial susceptibility by the agar dilution CLSI reference method to calculate MICs.ResultsOverall, 142 (35%) of the BV-associated vaginal isolates tested were resistant to metronidazole, 63 (15%) were resistant to clindamycin and 11 (2.6%) were resistant to rifaximin. Metronidazole resistance was observed most frequently amongGardnerella vaginalis(n = 100 isolates, 69% resistant),Atopobium vaginae(n = 62, 87% resistant) andMobiluncus(n = 40, 42% resistant), whereas most were susceptible to both clindamycin (197/202; 98% susceptible) and rifaximin (191/202; 95% susceptible). Both rifaximin and metronidazole were effective against all strains ofPrevotella bivia(n = 34),P timonensis(n = 33),Anaerococcus tetradius(n = 21), Finegoldia magna(n = 20) andPeptoniphilus lacrimalis(n = 20), whereas clindamycin resistance was detected among 74%, 42%, 19%, 30% and 30% of these anaerobes isolates, respectively. More than 90% ofPrevotella amnii(n = 33),Peptoniphilus harei(n = 23) andMegasphaera-like bacteria (n = 25) were susceptible to all three antibiotics. As expected, none of theLactobacillusisolates were susceptible to metronidazole, whereas a majority were susceptible to both clindamycin and rifaximinin vitro.ConclusionRifaximin had MIC values for a range of microorganisms associated with BV which were superior or similar to the other two drugs approved for the treatment of this condition and deserves clinical evaluation as a new therapeutic agent for the treatment of BV.

Evaluation of Caspofungin Susceptibility Testing by the New Vitek 2 AST-YS06 Yeast Card Using a Unique Collection of FKS Wild-Type and Hot Spot Mutant Isolates, Including the Five Most Common Candida Species

FKS mutant isolates associated with breakthrough or failure cases are emerging in clinical settings. Discrimination of these from wild-type (wt) isolates in a routine laboratory setting is complicated. We evaluated the ability of caspofungin MIC determination using the new Vitek 2 AST-Y06 yeast susceptibility card to correctly identify the fks mutants from wt isolates and compared the performance to those of the CLSI and EUCAST reference methods. A collection of 98 Candida isolates, including 31 fks hot spot mutants, were included. Performance was evaluated using the FKS genotype as the "gold standard" and compared to those of the CLSI and EUCAST methodologies. The categorical agreement for Vitek 2 was 93.9%, compared to 88.4% for the CLSI method and 98.7% for the EUCAST method. Vitek 2 misclassified 19.4% (6/31) of the fks mutant isolates as susceptible, in contrast to <4% for each of the reference methods. The overall essential agreement between the CLSI method and Vitek 2 MICs was 92.6% (88/95) but was substantially lower for fks mutant isolates (78.6% [22/28]). Correct discrimination between susceptible and intermediate Candida glabrata isolates was not possible, as the revised species-specific susceptibility breakpoint was not included in the Vitek 2 detection range (MIC of ≤0.250 to ≥4 mg/liter). In conclusion, the Vitek 2 allowed correct categorization of all wt isolates as susceptible. However, despite an acceptable categorical agreement, it failed to reliably classify isolates harboring fks hot spot mutations as intermediate or resistant, which was in part due to the fact that the detection range did not span the susceptibility breakpoint for C. glabrata.

Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Caspofungin Susceptibility Testing of Candida and Aspergillus Species

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for testing susceptibility to caspofungin of wild-type and fks mutant isolates of Candida and Aspergillus. Complete essential agreement was observed with the CLSI reference method, with categorical agreement for 94.1% of the Candida isolates tested. Thus, MALDI-TOF MS is a reliable and accurate method to detect fungal isolates with reduced caspofungin susceptibility.

Evaluation of the MicroScan MICroSTREP Plus Antimicrobial Panel for Testing ß-Hemolytic Streptococci and Viridans Group Streptococci

BackgroundIn order to determine the clinical usefulness of the MicroScan (Siemens Healthcare Diagnostics, USA) MICroSTREP plus antimicrobial panel (MICroSTREP) for testing antimicrobial susceptibility of β-hemolytic streptococci (BHS) and viridans group streptococci (VGS), we compared the accuracy of MICroSTREP with that of the CLSI reference method.MethodsSeventy-five BHS and 59 VGS isolates were tested for antimicrobial susceptibility to ampicillin, penicillin, cefotaxime, meropenem, erythromycin, clindamycin, levofloxacin, and vancomycin by using MICroSTREP and the CLSI agar dilution method.ResultsThe overall essential agreement with regard to minimum inhibitory concentrations (MICs) (within ±1 double dilution) between MICroSTREP and the CLSI reference method was 98.2%, and categorical agreement (CA) was 96.9%. For the BHS isolates, the CA for erythromycin was 96.0%, whereas that for cefotaxime, meropenem, levofloxacin, and vancomycin (for ampicillin, penicillin, and clindamycin; 98.7%) was 100%. For the VGS isolates, the CA for penicillin was 84.7% and that for erythromycin, clindamycin, and vancomycin (for meropenem, 86.5%; for ampicillin, 88.1%; and for cefotaxime and levofloxacin, 96.6%) was 100%. All categorical errors of penicillin and ampicillin in the VGS isolates were minor.ConclusionsThe accuracy of MICroSTREP is comparable to that of the CLSI reference method, suggesting that this panel can be effective for testing antimicrobial susceptibility of BHS and VGS.