WalkAway System Research Articles

In Vitro Synergistic Activity of Ceftazidime-Avibactam and Aztreonam against New Delhi Metallo-β-Lactamase-Producing Clinical Enterobacterales Isolates

Background: Carbapenemase-producing-Enterobacterales (CPE) infections are on the rise and associated with increased morbidity and mortality, due to a limited number of therapeutic options. The goal of this study was to assess the synergistic activity of ceftazidime-avibactam (CZA) and aztreonam (ATM) combination against phenotypically and genetically characterized blaMBL-producing Enterobacterales.Methods: In this study, forty (n=40) non-repeat, CPE clinical isolates, including: n=35 Klebsiella pneumoniae, n=2 each of Escherichia coli and Klebsiella oxytoca, and n=1 Enterobacter cloacae isolates were identified and susceptibilities were assessed using the Vitek-II compact (bioMérieux, Inc., France) and Microscan Walkaway (Beckman Coulter) systems. Genotypic analysis of clinically relevant carbapenemases was performed by using Xpert-Carba-R assay on GeneXpert (Cepheid, USA). The minimum inhibitory concentrations (MICs) and synergy testing of CZA and ATM, were tested by the Etest fixed ratio method (bioMérieux, Inc., France). The fractional inhibitory concentration index (FICI) was calculated for each antibiotic.Results: Our results showed that 97.5% of blaMBL-producing Enterobacterales isolates were susceptible to an in-vitro CZA + ATM combination regimen. The fractional inhibitory concentration index (FICI) ranged from 0.001 to 1.001. Among the tested CPE isolates, Synergy was observed in 36/40 (90%), an additive effect was observed in 5% (n=2)’ while two (5%) isolates showed indifference. There was no antagonism observed in our study.Conclusion: Our study exhibited a potent activity of CZA and ATM combination synergy against clinical CPE metallo- β-lactamase producers. More extensive studies involving a variety of Gram-negative pathogens with different resistance mechanisms are required to determine the efficacy of this combination regimen.Keywords: Synergy; Aztreonam; Ceftazidime-Avibactam; Carbapenem-Resistant Enterobacterales; Etest

Bacterial and Genetic Features of Raw Retail Pork Meat: Integrative Analysis of Antibiotic Susceptibility, Whole-Genome Sequencing, and Metagenomics.

The global antibiotic resistance crisis, driven by overuse and misuse of antibiotics, is multifaceted. This study aimed to assess the microbiological and genetic characteristics of raw retail pork meat through various methods, including the isolation, antibiotic susceptibility testing (AST), whole-genome sequencing (WGS) of selected indicator bacteria, antibiotic residue testing, and metagenomic sequencing. Samples were purchased from 10 pre-selected retail stores in Gauteng, South Africa. The samples were aseptically separated, with portions sent to an external laboratory for isolating indicator bacteria and testing for antibiotic residues. Identification of the isolated bacteria was reconfirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). AST was performed using the Microscan Walkaway system (Beckman Coulter, Brea, CA, USA). WGS and metagenomic sequencing were performed using the Illumina NextSeq 550 instrument (San Diego, CA, USA). The isolated E. coli and E. faecalis exhibited minimal phenotypic resistance, with WGS revealing the presence of tetracycline resistance genes. Both the isolated bacteria and meat samples harboured tetracycline resistance genes and the antibiotic residue concentrations were within acceptable limits for human consumption. In the metagenomic context, most identified bacteria were of food/meat spoilage and environmental origin. The resistome analysis primarily indicated beta-lactam, tetracycline and multidrug resistance genes. Further research is needed to understand the broader implications of these findings on environmental health and antibiotic resistance.

Preliminary reading of antibiogram by microdilution for clinical isolates in urine culture.

We evaluated a modification of automated antibiograms in urine cultures designed to facilitate the early interpretation of minimum inhibitory concentrations (MICs) and accelerate the targeted treatment of urinary tract infections (UTIs), METHODS: A prospective study was conducted of 309 isolates (219 Enterobacteriaceae, 75 Enterococcus spp., and 15 non-fermenting Gram-negative bacilli (NFGNB), and a retrospective study of 9 carbapenemase-producing clinical isolates from urine cultures. Colonies grown on conventional isolation plates were inoculated in MicroScan Walkaway system panels and incubated for 7 h, using a MicroScan AutoScan-4 plate reader for preliminary MIC determination by turbidimetry. Resulting antibiograms were compared with definitive antibiograms obtained after incubation for 17 h. Preliminary and definitive readings were concordant for 86.7% of Gram-positive cocci isolates (65/75), 61.6% of Enterobacteriaceae (135/219), and 53.3% of NFGNB. The agreementrate was greater than 90% for most antimicrobials against Gram-positive cocci (94.7% or more) and Enterobacteriaceae, (97.2% or more for 10 of 17 antibiotics) except with nitrofurantoin (89%). The agreement rate was 86.7% or more for most antibiotics against NFGNB apart from piperacillin/tazobactam, aztreonam, amikacin, and ciprofloxacin. Gram-negative bacilli showed the highest differences in MIC values between preliminary and definitive readings. A preliminary antibiogram reading may be useful in urine cultures to reduce the delay before targeted antibiotherapy, especially against Enterobacteriaceae and Gram-positive cocci, but not in cases of carbapenemase-producing NFGNB. Further local studies are warranted to evaluate the usefulness of this approach in relation to resistance rates.

Rapid AST: Possibility of inferring resistance mechanisms with complex phenotypes.

The new automated systems designed for rapid performance of AST have significantly reduced the response time for susceptibility testing of microorganisms causing bacteremia and sepsis. The Accelerate Pheno® system (AAC) is one such system. Our objective for this study was to determine whether the AAC system is capable of providing an accurate susceptibility profile to infer resistance mechanisms in different carbapenemase-producing isolates when compared to the MicroScan WalkAway System (MWS). Disk diffusion method was also performed on all isolates as a reference method. Additionally, we compared the results obtained with the routine AST production system. We selected 19 isolates from the cryobank of the Microbiology department, all of which were carbapenemase-producing gram-negative bacilli. AAC was able to identify and infer the resistance of a total of 10 isolates, with an EA and CA of 84.2% for meropenem and 88.2% and 64.7% for ertapenem EA and CA, respectively. If we consider the disk diffusion technique, the CA was 57.9% and 76.5% for meropenem and ertapenem. However, in the presence of carbapenemases, AAC was not able to provide adequate MICs or infer the resistance mechanisms of the isolates accurately. Further studies with a larger number of isolates, including the new antibiotics ceftolozane/tazobactam and ceftazidime/avibactam, are needed for a more comprehensive comparison.

A-273 Multicenter Assessment of the Accuracy of MIC Results for Piperacillin/Tazobactam with MicroScan Dried Gram-Negative MIC Panels using CLSI Breakpoints

Abstract Background MIC data from MicroScan Dried Gram-negative MIC (MSDGN) Panels with piperacillin/tazobactam were evaluated with CLSI-M100-ED32 breakpoints for Acinetobacter, Enterobacterales, Other-Non-Enterobacterales, and proposed CLSI breakpoints for Pseudomonas aeruginosa from a multicenter clinical study. MIC results were compared to results obtained with CLSI frozen broth microdilution panels. Materials/Methods The study included a total of 683 clinical isolates tested using Prompt and turbidity during efficacy and challenge phases. MSDGN panels were evaluated at three clinical sites, comparing MIC values obtained using MSDGN panels to MICs utilizing CLSI reference panel. MSDGN panels were incubated at 35 ± 1°C and read on WalkAway System, autoSCAN-4 instrument, and read visually at 16–20 h. Frozen reference panels were prepared according to CLSI methodology, incubated for 16–20 h for Enterobacterales, Other Non-Enterobacterales, and Pseudomonas aeruginosa and for 20–24 h for Acinetobacter and read visually. CLSI breakpoints(mg/L) used for interpretation of MIC results were: ≤16/4 S, 32/4–64/4 I, ≥128/4 R for Acinetobacter and Other Non Enterobacterales, ≤8/4 S, 16/4 SDD, ≥32/4 R for Enterobacterales, and ≤16/4 S, 32/4 I, >64/4 R for Pseudomonas aeruginosa. Results When compared to frozen reference panel results, essential and categorical agreements for all isolates tested are as follows with these recommendations: Due to elevated major error rates for S. marcescens and WalkAway reads with Prompt, results should be confirmed visually prior to reporting. Due to performance with P. rettgeri and S. liquefaciens complex, do not report drug, therapy, or MIC. Conclusion Piperacillin/tazobactam MIC results for gram-negative clinical isolates obtained with the MSDGN panel correlate with MICs obtained using frozen reference panels with updated CLSI interpretive criteria.

A-274 Validation of Cefepime-Taniborbactam MIC Antimicrobial Susceptibility Test for MicroScan Dried Gram-Negative MIC Panels from a Multicenter Assessment of Enterobacterales and Pseudomonas aeruginosa

Abstract Background Cefepime-taniborbactam is an investigational agent with activity against carbapenem and multidrug-resistant Enterobacterales and Pseudomonas aeruginosa.1 Given the need for accurate antimicrobial susceptibility testing results to support patient care decisions, a multicenter study was conducted with Enterobacterales and Pseudomonas aeruginosa clinical isolates on investigational MicroScan Dried Gram-negative MIC (MSDGN) panels with cefepime-taniborbactam.2 Materials/Methods MSDGN panels were evaluated at three clinical sites by comparing MIC values obtained using MSDGN panels to MICs utilizing CLSI broth microdilution reference panels. The study included 490/491 Enterobacterales and 65 Pseudomonas aeruginosa clinical isolates tested using Prompt® and turbidity methods of inoculation during the combined efficacy and challenge phases. MSDGN panels were incubated at 35 ± 1°C and read on the WalkAway System, the autoSCAN-4 instrument, and visually at 16–20 h. Frozen reference panels were prepared according to CLSI/ISO methodology, incubated for 16–20 h and read visually. Results Essential agreement was calculated compared to MIC results from frozen reference panels for isolates tested in efficacy and challenge and found in the following table. Conclusion Cefepime-taniborbactam MIC results for Enterobacterales and Pseudomonas aeruginosa obtained with the MSDGN panel correlate well with MICs obtained using frozen reference panels in this multicenter study.Pending approval by the US Food and Drug Administration; not available for in vitro diagnostic use in the US. Not for Distribution in the US.Pending submission and clearance by the US Food and Drug Administration; not yet available for in vitro diagnostic use in the US.For Investigational Use Only. Performance characteristics of this product have not been established.Read methodEnterobacterales Essential agreement (EA) % (n/N)Pseudomonas aeruginosaEssential agreement (EA) % (n/N)PromptTurbidityPromptTurbidityWalkAway90.0 (441/490)99.2 (487/491)100.0 (65/65)100.0 (65/65)autoSCAN-494.3 (462/490)99.0 (486/491)98.5 (64/65)98.5 (64/65)Visually92.7 (454/490)99.2 (487/491)100.0 (65/65)100.0 (65/65)Abbreviations: Prompt, Prompt inoculation method, Turbidity, Turbidity inoculation method.

Development and validation of a fully automated laboratory-developed test for detection of HSV-1/2 and VZV in clinical samples run on the Panther Fusion® system

The purpose of this study was to develop a laboratory developed test (LDT) for HSV1/2 and VZV to run on fully automated Hologic Panther Fusion® System. The Panther Fusion System is a fully automated walkaway system, providing end-to-end workflow from sample input to DNA/RNA extraction, amplification, automated analysis, and reporting to a laboratory information system (LIS). The LDT was developed and validated on 230 clinical and 20 reference samples (n = 250) and compared to a commercially available kit. Assessment of the analytical and diagnostic performances of the LDT revealed >98% accuracy, sensitivity, and specificity, which is consistent with or better than many of the commercial or laboratory-developed tests available. The advantage of this LDT is that it is designed to perform a single-run full female health screening in parallel with 4 commercially available Hologic kits for Chlamydia trachomatis/Neisseria gonorrhea (CT/NG), Trichomonas vaginalis (TV), Mycoplasma genitalium (MG), and bacterial vaginosis (BV).

230. Multicenter Assessment of the Accuracy of MIC Results for Colistin with MicroScan Dried Gram Negative MIC Panels using CLSI Breakpoints

Abstract Background A multicenter study was performed to evaluate the accuracy of MIC results for colistin on a MicroScan Dried Gram Negative MIC (MSDGN) Panel when compared to results obtained with frozen broth microdilution panels prepared according to CLSI/EUCAST methodology. Note: Colistin is not available in all countries. Methods MSDGN panels were evaluated at three clinical sites (including Beckman Coulter) by comparing MIC values obtained using the MSDGN panels to MICs obtained utilizing a CLSI/EUCAST broth microdilution reference panel. The study included 407 clinical isolates tested using the turbidity and Prompt® methods of inoculation. MSDGN panels were incubated in the WalkAway System (35 ± 1°C) and read on the WalkAway System, the autoSCAN-4 instrument, and read visually at 16-20 hours. Frozen reference panels were prepared and inoculated according to CLSI/EUCAST Joint Working Group Recommendations for MIC determination of Colistin (Polymyxin E) and incubated for 16-20 hours and read visually. CLSI M100 ED32 breakpoints (µg/mL) used for interpretation of MIC results were: Enterobacterales ≤ 2 I, ≥ 4 R; P. aeruginosa ≤ 2 I, ≥ 4 R. Results Essential, categorical agreement, and categorical errors were calculated using MIC results from WalkAway reads for MSDGN panels in Table 1. All other read methods yielded similar results. Categorical agreement was not calculated for Acinetobacter spp. as MIC only is reported. Table 1- Results Conclusion This multicenter study showed that colistin MIC results for Gram Negative bacteria obtained with the MSDGN panel correlate well with MICs obtained using frozen reference panels with CLSI interpretive criteria. © 2022 Beckman Coulter. All rights reserved. All other trademarks are the property of their respective owners. Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the U.S. and other countries. Disclosures Stefan Riedel, M.D., PhD., D(ABMM), FCAP, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc. Maria Traczewski, BS, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc. Denise Beasley, BS, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc. Regina Brookman, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter Jose Diaz, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter Zabrina Lockett, PhD, Beckman Coulter, Inc.: Employee of Beckman Coulter Jennifer Chau, PhD, Beckman Coulter, Inc.: Employee of Beckman Coulter.

342. Multicenter Evaluation of Fosfomycin MIC Results for Enterobacterales Using EUCAST V11 Breakpoints (i.v. and oral for uncomplicated UTI only, E. coli) on MicroScan Dried Gram Negative MIC Panels

Abstract Background EUCAST V11 fosfomycin breakpoints for Enterobacterales (i.v. and oral for uncomplicated UTI only, E. coli) were evaluated against data from a multicenter clinical study on a MicroScan Dried Gram Negative MIC (MSDGN) Panel. MIC results were compared to results obtained with agar dilution reference prepared according to CLSI methodology. Methods A total of 344 Gram negative clinical isolates including 191 Enterobacterales were tested using the turbidity and Prompt® methods of inoculation during the efficacy phase at three clinical sites. An evaluation was conducted by comparing MIC values obtained using the MSDGN panels to MICs utilizing the CLSI agar dilution reference. MSDGN panels were incubated at 35 ± 1°C and read on the WalkAway System, the autoSCAN-4 instrument, and read visually at 16-20 hours. Agar dilution plates were prepared according to CLSI methodology, incubated for 16-20 hours and read visually. EUCAST v11.0 fosfomycin breakpoints (mg/L) used for interpretation of MIC results were: i.v. for Enterobacterales ≤ 32 S, > 32 R and oral for uncomplicated UTI only, E. coli ≤ 8 S, > 8 R. Results Essential agreement, categorical agreement and categorical errors were calculated compared to MIC results from agar dilution plates. Results for all efficacy isolates with turbidity inoculation and manual read are found in Table 1. Table 1- Results Conclusion This multicenter study showed that fosfomycin MIC results for i.v. for Enterobacterales and E. coli (oral, for uncomplicated UTI only) obtained with the MSDGN panel correlate well with MICs obtained using CLSI agar dilution reference using EUCAST interpretive criteria. © 2022 Beckman Coulter. All rights reserved. All other trademarks are the property of their respective owners. Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. Disclosures Omai Garner, PhD, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc. Amanda Harrington, PhD, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc.|bioMeriuex/BioFire: Grant/Research Support Sharon DesJarlais, BS, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc. Maria Traczewski, BS, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc. Denise Beasley, BS, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc. Jose Diaz, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter Regina Brookman, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter Zabrina Lockett, PhD, Beckman Coulter, Inc.: Employee of Beckman Coulter Jennifer Chau, PhD, Beckman Coulter, Inc.: Employee of Beckman Coulter.

520. Development of Cefepime-taniborbactam MIC Antimicrobial Susceptibility Test (AST) for Enterobacterales and Pseudomonas aeruginosa on MicroScan Dried Gram-negative MIC Panels

Abstract Background Automated AST devices are critical to inform appropriate care of patients with bacterial infections. Cefepime-taniborbactam is an investigational agent under development to treat infections due to multidrug-resistant bacteria, including carbapenem-resistant Enterobacterales and Pseudomonas aeruginosa. Development of a cefepime-taniborbactam antimicrobial susceptibility test was completed for the MicroScan Dried Gram-negative MIC (MSDGN) Panel when compared to CLSI broth microdilution reference panels. Methods Development was conducted by comparing MICs obtained using the MSDGN panel to MICs using a CLSI broth microdilution reference panel. The concentration range of cefepime-taniborbactam evaluated was 0.12/4-64/4 µg/mL. A total of 1376 isolates (1256 Enterobacterales and 120 P. aeruginosa) were tested at 16, 18, and 20 hours incubation times (for visual read and autoSCAN-4). A total of 917 isolates (837 Enterobacterales and 80 P. aeruginosa) were tested at 16 and 18 hours incubation time for the WalkAway System. All isolates were tested using turbidity and Prompt® methods of inoculation. MSDGN panels were incubated at 35 ± 1°C. Reference panels, prepared according to ISO methodology, were inoculated using the turbidity inoculation method. All frozen reference panels were incubated at 35 ± 2°C and read visually. Rates of essential agreement (MicroScan MIC within ± 1 doubling dilution of reference MIC) were determined for each combination of inoculation method and read method. Results Essential agreement rates ranged from 93.0% to 98.8% for all isolates tested during development. Essential agreement rates for each inoculation and read method are presented in Table 1. Table 1- Results Conclusion Cefepime-taniborbactam MIC results obtained with the MSDGN panel correlated well with MIC results obtained using reference panels. Rates of essential agreement were ≥93.0% for all inoculation and read methods. These development data support the continued evaluation of MSDGN panel with cefepime-taniborbactam in a multicenter trial. Pending submission and clearance by the United States Food and Drug Administration; not yet available for in vitro diagnostic use in the US. For Investigational Use Only. The performance characteristics of this product have not been established. Disclosures Enrique J. Fernandez, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter Robert L. Williams, PhD, Beckman Coulter, Inc.: Employee of Beckman Coulter Vasna Carr, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter Vasna Carr, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter Miller Renae, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter.

Laboratory Diagnostic Methods and Antibiotic Resistance Patterns of Staphylococcus aureus and Escherichia coli Strains: An Evolving Human Health Challenge.

Raw ground meat is known as a transmission vehicle for biological agents that may be harmful to human health. The objective of the present study was to assess microbiological quality of the ground meats. A total of 280 samples of local and imported chilled meats were randomly collected from retail shops in Buraydah City, Saudi Arabia. The meat samples were microbiologically analyzed using standard methods, peptide mass fingerprinting (PMF) technique, MicroScan Walkaway System (MicroScan) and qPCR System. The imported meat was more bacterially contaminated than local meat, with variable contamination degrees of Staphylococcus aureus (40.33%), Escherichia coli (36.13%), Hafnia alvei (7.56%), Pseudomonas spp. (6.72%), Salmonella spp. (5.88%) and Aeromonas spp. (3.36%). PMF verified all the isolated bacteria by 100%, compared to 75-95% achieved by MicroScan. The gene encoding flagellin (fliC) was recognized in 67.44% of E. coli strains, while the thermonuclease (nuc) and methicillin resistance (mecA) genes were detected in 100% S. aureus and 39.6% of methicillin-resistant S. aureus (MRSA) strains, respectively. The S. aureus and E. coli strains were highly resistant to multiple antibiotics (e.g., ampicillin, amoxicillin-clavulanic acid and cephalothin). For identifying various foodborne pathogens, PMF has been recognized as a powerful and precise analytical method. In light of the increasing use of PMF to detect multidrug-resistant bacteria, this study emphasizes the need for improved ways of treating and preventing pathogens, as well as setting up monitoring systems to guarantee hygiene and safety in meat production.

P339 A study of environmental risk factors in chronic rhinosinusitis

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesChronic rhinosinusitis (CRS) is a complex disease that incorporates many different conditions. Our aim is to determine the role of environmental risk factors in chronic rhinosinusitis.MethodsThis was a case-control study where forty cases of CRS in the age group of 18-62 years were recruited from the ENT-OPD of UCMS and GTB Hospital, Delhi, a tertiary care hospital, along with 40 age-appropriate healthy controls. Informed and written consent was obtained from all the subjects. Detailed history and examination along with routine blood and radiological investigations were done. Subjective assessment of disease was done using visual analog scales of SNOT 22 questionnaire, environmental questionnaire, and saccharine test. Objective assessment was done using endoscopic grading according to Lund and Kennedy scoring. CT scores were also obtained using Lund and Mackay staging system. Passive air sampling was done by the settle plate method and Anderson six-stage cascade impactor was used for active air sampling. Blood agar and Sabouraud dextrose agar plates were inserted on the six stages of the cascade impactor, which were later incubated at 35°C and 25°C respectively, for 18-24 hours. Bioaerosol concentrations were calculated and MicroScan WalkAway system was used to identify bacterial and fungal growth. The correlation between total bioaerosol concentration and the symptom score was done by spearman's correlation coefficient; a comparison of mean total bioaerosol concentration between the case and control groups was done by Mann-Whitney U-test.ResultsThe mean total bio-aerosol concentration from active air sampling in the case group was 458.52 CFU/m3 and 437.63 CFU/m3 in control, which was not significant. It was found to be negatively correlated, but not significant with various parameters in case groups like SNOT 22 score, environmental exposure score, saccharine test, smoking, and endoscopic score. A positive but insignificant correlation was found between total bioaerosol concentration and absolute eosinophilic count and CT score. The mean CFU/100 mm3 counted after passive air sampling between case and control group was 34.03 and 30.88 CFU/100mm3 which was not significant. Fungal growths were mostly Aspergillus flavus and A. Niger followed by Cladosporium spp.ConclusionThe fungal burden was high in all the households of the case as well as control groups, although, a significant correlation could not be established. The environmental conditions found in this study were found to be conducive to future risk of exacerbation of preexisting fungal infections for which, indoor air quality needs to be monitored.

P444 Detection of causative agents of infectious keratitis in patients from western rajasthan

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectiveTo determine the spectrum of causative agents, the related risk factors, and their association in patients of infectious keratitis.MethodologyIt was a prospective study conducted over a period of 18 months from August 2018 to January 2020, which included 100 patients attending the Ophthalmology OPD with features of keratitis. Ophthalmological examination was followed by corneal scrapings’ collection, which were subjected to culture, microscopy, and molecular diagnostic tools. Bacterial isolates were identified by conventional methods and MicroScan Walkaway system while the isolated fungi were identified conventionally. Pan-fungal primers were used to detect fungal elements directly from the sample.ResultsOut of 100, 41 cases were positive by culture, of which 32 (78.04%) had fungal and nine (21.95%) had bacterial keratitis. Fusarium spp. accounted for 33.33% of fungal and Pseudomonas aeruginosa accounted for 55.55% of the bacterial isolates. Fungal material was detected in 41% using pan fungal primers. Cases were maximally recorded during July-October. Traumatic history was present in 78% patients caused by vegetative matter (49%). A male preponderance (67%) was also observed. Four patients underwent evisceration in spite-of rigorous management.ConclusionPoor prognosis emphasizes the need for faster diagnostics, which can detect the causative agents from the clinical specimen itself, reinforcing the concept of clinical metagenomics.

Acinetobacter baumannii complex, national laboratory-based surveillance in South Africa, 2017 to 2019.

ObjectiveWe aimed to provide an analysis of A. baumannii complex (ABC) isolated from blood cultures in South Africa.Materials and methodsABC surveillance was conducted from 1 April 2017 to 30 September 2019 at 19 hospital sites from blood cultures of any age and sex. Organism identification was performed using the MALDI-TOF MS and antimicrobial susceptibility testing (AST), MicroScan Walkaway System. We confirmed colistin resistance with Sensititre, FRCOL panel, and selected for whole-genome sequencing.ResultsDuring the study period, we identified 4822 cases of ABC, of which 2152 cases were from 19 enhanced surveillance sites were reported during the enhanced surveillance period (1 August 2018 to 30 September 2019). Males accounted for 54% (2611/4822). Of the cases with known age, 41% (1968/4822) were infants (< 1-year-old). Seventy-eight percent (1688/2152) of cases had a known hospital outcome, of which 36% (602/1688) died. HIV status was known for 69% (1168/1688) of cases, and 14% (238/1688) were positive. Eighty-two percent (1389/1688) received antimicrobial treatment in admission. Three percent (35/1389) of cases received single colistin. Four percent (75/2033) were resistant to colistin. At least 75% of the isolates (1530/2033) can be classified as extensively drug-resistant (XDR), with resistance to most antibiotics except for colistin. The majority, 83% (20/24), of the colistin-resistant isolates were of the sequence type (ST) 1. Resistance genes, both plasmid- and chromosomal- mediated were not observed. Although all isolates had, nine efflux pump genes related to antimicrobial resistance.ConclusionOur surveillance data contributed to a better understanding of the natural course of A. baumannii disease, the patient characteristics among infants, and the level of resistance. At least two-thirds of the isolates were extensively drug-resistant, and four percent of isolates were resistant to colistin.

1079. Development of Tebipenem MIC Antimicrobial Susceptibility Test for Gram-negative Bacteria on MicroScan Dried Gram-negative MIC Panels

Abstract Background Development of a tebipenem antimicrobial susceptibility test was completed for the MicroScan Dried Gram-negative MIC (MSDGN) Panel when compared to CLSI broth microdilution reference panels. Methods Development was conducted by comparing MICs obtained using the MSDGN panel to MICs using a CLSI broth microdilution reference panel. A total of 669 Enterobacterales isolates were tested at 16, 18, and 20 hour incubation times using the turbidity and Prompt®* methods of inoculation. MSDGN panels were incubated at 35 ± 2ºC and read on the WalkAway System, the autoSCAN-4 instrument, and read visually. Frozen reference panels, prepared according to ISO/CLSI methodology, were inoculated using the turbidity inoculation method. All frozen reference panels were incubated at 35 ± 2ºC and read visually. Dilution sequence evaluated is 0.03-32 µg/mL. Results When compared to frozen reference panel results, essential agreement for all isolates tested during development are as follows: Conclusion The development data showed that tebipenem MIC results for Enterobacterales obtained with the MSDGN panel correlate well with MICs obtained using frozen reference panels. Essential agreement is &amp;gt; 90% for all inoculation and read methods. For Investigational Use Only. The performance characteristics of this product have not been established. * Prompt® is a registered trademark of 3M Company, St. Paul, MN USA. © 2021 Beckman Coulter. All rights reserved. Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. Disclosures Jose Enrique Fernandez, n/a, Beckman Coulter (Employee) Vasna Carr, n/a, Beckman Coulter (Employee) Renae Miller, n/a, Beckman Coulter (Employee)

GC-mass determination for the biodegradative products of 2,6-dimethylpyridine using Dead-Sea bacterial isolate

Dead sea soil is known for its hypersaline environment and it is a promising location for isolating extremophilic bacteria with interesting metabolic features. In the current study, we isolated a gram-positive bacterium with the ability to degrade 2,6-dimethyl pyridine (2,6-DMP), also known as 2,6-lutidine, a chemical pollutant. The isolated bacteria were identified using the automated Microscan Walkaway system and the different biochemical reactions were determined. In minimal media using the 2,6-DMP as a sole carbon source, the bacterial isolate showed the ability to convert approximately 40 % of 2,6-DMP within 5 days. The GC-Mass analysis for the degradation products indicated that mono- and dihydroxylation for the pyridine ring and oxidation of one or both of the terminal methyl groups have occurred. Based on this finding, this isolated bacterial can further be utilized for bioremediation purposes.

673. Updated CLSI Ciprofloxacin Breakpoints from a Multicenter Assessment for Enterobacterales, Salmonella spp. and Pseudomonas aeruginosa Using MicroScan Dried Gram Negative MIC Panels

BackgroundUpdated US FDA/CLSI ciprofloxacin breakpoints were evaluated against data from a multicenter clinical study with Enterobacterales, Salmonella spp. and P. aeruginosa on a MicroScan Dried Gram-negative MIC (MSDGN) Panel. MIC results were compared to results obtained with frozen broth microdilution panels prepared according to CLSI methodology.MethodsMSDGN panels were evaluated at three clinical sites by comparing MIC values obtained using the MSDGN panels to MICs utilizing a CLSI broth microdilution reference panel. Data from the combined phases of efficacy and challenge included 803 Enterobacterales, Salmonella spp. and P. aeruginosa clinical isolates tested using the turbidity and Prompt® methods of inoculation. To demonstrate reproducibility, a subset of 12 organisms were tested on MSDGN panels at each site during reproducibility. MSDGN panels were incubated at 35 ± 1ºC and read on the WalkAway System, the autoSCAN-4 instrument, and visually. Read times for the MSDGN panels were at 16-20 hours. Frozen reference panels were prepared and read according to CLSI methodology. FDA and CLSI breakpoints (µg/mL) used for interpretation of MIC results were: Enterobacterales ≤ 0.25 S, 0.5 I, ≥ 1 R; Salmonella spp. ≤ 0.06 S, 0.12-0.5 I, ≥ 1 R; P. aeruginosa ≤ 0.5 S, 1 I, ≥ 2 R.ResultsEssential and categorical agreement was calculated compared to frozen reference panel results. Results for isolates tested during efficacy and challenge with Prompt inoculation and manual read are as follows: Read Method Essential Agreement (EA) % Categorical Agreement (CA) % Very Major Error (VMJ) % Major Error (MAJ) % Enterobacterales 93.8(646/689)97.7(673/689)0.0(0/146)0.0(0/535) Salmonella spp. 100(21/21)95.2(20/21)0.0(0/1)0.0(0/18) P. aeruginosa 94.6(88/93)91.4(85/93)0.0(0/29)1.7(1/58)ConclusionCiprofloxacin MIC results for Enterobacterales, Salmonella spp., and P. aeruginosa obtained with the MSDGN panel correlate well with MICs obtained using frozen reference panels using updated FDA/CLSI interpretive criteria in this multicenter study.* PROMPT® is a registered trademark of 3M Company, St. Paul, MN USA.BEC, the stylized logo and the BEC product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the US and other countries.Disclosures Maria M. Traczewski, BS MT (ASCP), Beckman Coulter (Scientific Research Study Investigator) Denise Beasley, BS, Beckman Coulter (Other Financial or Material Support, Research personnel) Amanda Harrington, PhD, Beckman Coulter (Scientific Research Study Investigator) Sharon DesJarlais, BS, Beckman Coulter (Other Financial or Material Support, Research personnel) Omai Garner, PhD, D(ABMM), Beckman Coulter (Scientific Research Study Investigator) Christine Hastey, PhD, Beckman Coulter (Employee) Regina Brookman, BS, Beckman Coulter (Employee) Zabrina Lockett, MS, Beckman Coulter (Employee) Jennifer Chau, PhD, Beckman Coulter (Employee)

667. Multicenter Assessment of Enterobacterales, Salmonella spp. and Pseudomonas aeruginosa Using Updated CLSI Levofloxacin Breakpoints on MicroScan Dried Gram Negative MIC Panels

BackgroundData from a multicenter clinical study with Enterobacterales, Salmonella spp. and P. aeruginosa on a MicroScan Dried Gram-negative MIC (MSDGN) Panel was evaluated with updated US FDA/CLSI levofloxacin breakpoints. MIC results were compared to results obtained with frozen broth microdilution panels prepared according to CLSI methodology.MethodsA total of 839 Enterobacterales, Salmonella spp. and P. aeruginosa clinical isolates were tested at three clinical sites in efficacy and challenge combined. MSDGN panels were evaluated with turbidity and Prompt® methods of inoculation. MIC values obtained from the MSDGN panels were compared to MICs utilizing a CLSI broth microdilution reference panel. To assess reproducibility, a subset of 15 organisms were tested on MSDGN panels at each site. MSDGN panels were incubated at 35 ± 1ºC and read on the WalkAway System, the autoSCAN-4 instrument, and visually. Read times for the MSDGN panels were at 16-20 hours. Frozen reference panels were prepared and read according to CLSI methodology. FDA and CLSI breakpoints (µg/mL) used for interpretation of MIC results were: Enterobacterales ≤ 0. 5 S, 1 I, ≥ 2 R; Salmonella spp. ≤ 0.12 S, 0.25-1 I, ≥ 2 R; P. aeruginosa ≤ 1 S, 2 I, ≥ 4 R.ResultsEssential and categorical agreement were calculated compared to frozen reference panel results. Results for isolates tested during efficacy and challenge with Prompt inoculation and manual read are as follows: Read Method Essential Agreement (EA) % Categorical Agreement (CA) % Very Major Error (VMJ) % Major Error (MAJ) % Enterobacterales 96.2(638/663)96.7(641/663)1.5(2/133)0.0(0/515) Salmonella spp. 100(83/83)98.8(82/83)0.0(0/19)0.0(0/38) P. aeruginosa 94.6(88/93)93.6(87/93)0.0(0/34)1.9(1/54)ConclusionLevofloxacin MIC results for Enterobacterales, Salmonella spp., and P. aeruginosa obtained with the MSDGN panel correlate well with MICs obtained using frozen reference panels using updated FDA/CLSI interpretive criteria in this multicenter study.* PROMPT® is a registered trademark of 3M Company, St. Paul, MN USA.BEC, the stylized logo and the BEC product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the US and other countries.Disclosures Omai Garner, PhD, D(ABMM), Beckman Coulter (Scientific Research Study Investigator) Maria M. Traczewski, BS MT (ASCP), Beckman Coulter (Scientific Research Study Investigator) Denise Beasley, BS, Beckman Coulter (Other Financial or Material Support, Research personnel) Amanda Harrington, PhD, Beckman Coulter (Scientific Research Study Investigator) Sharon DesJarlais, BS, Beckman Coulter (Other Financial or Material Support, Research personnel) Christine Hastey, PhD, Beckman Coulter (Employee) Regina Brookman, BS, Beckman Coulter (Employee) Zabrina Lockett, MS, Beckman Coulter (Employee) Jennifer Chau, PhD, Beckman Coulter (Employee)

1267. Comparative activity of omadacycline against extended-spectrum beta-lactamase positive and negative Escherichia coli and Klebsiella pneumoniae strains recovered from urine specimens

BackgroundOmadacycline (OMC) is a novel tetracycline (TET) derivative antibiotic with activity against TET-resistant Enterobacterales. OMC is available in both oral and intravenous formulations and is has been studied as a treatment of uncomplicated urinary tract infection (UTI) and acute pyelonephritis. The purpose of this study was to evaluate OMC activity against extended-spectrum beta-lactamase (ESBL) positive and negative Enterobacterales strains recovered from urine specimens.MethodsUrine samples from patients with suspected UTI were quantitatively plated onto blood agar and MacConkey agar plates in the microbiology lab of Wake Forest Baptist Medical Center. After overnight incubation, colonies were identified to the species level by MALDI-TOF system. Susceptibility testing was performed for isolates of E. coli and K. pneumoniae. OMC and TET susceptibility testing was performed by disk diffusion and gradient strip methodologies. Results were interpreted in accordance with the Clinical and Laboratory Standards Institute (CLSI) or Food and Drug Administration breakpoints. Isolates were tested in triplicate. ESBL screening and susceptibility testing to oral antibiotics commonly prescribed for UTI were performed by the MicroScan WalkAway System. Susceptibility rates and MIC50/90 were calculated and subsets of isolates were analyzed using descriptive statistics.ResultsA total of 204 isolates, including 102 E. coli and 102 K. pneumoniae, were tested. All but 1 isolate (99.5%) exhibited categorical agreement in results generated by the strip (Table 1) and disk (data not shown) methods and this was considered a minor error involving an intermediate result. OMC MIC90 for E. coli and K. pneumoniae were 6 µg/mL and >32 µg/mL, respectively. OMC displayed increased susceptibility rates compared to TET regardless of isolate species or ESBL positivity (Table 2).Table 1. Omadacycline Minimum Inhibitory Concentrations (MICs, µg/mL) Table 2. Susceptibilities of Oral Antibiotics Used to Treat UTI (% S) ConclusionOMC exhibits promising antimicrobial activity against TET-resistant and ESBL-positive E. coli and K. pneumoniae. OMC displays superior activity to ESBL positive E. coli when compared to ESBL positive K. pneumoniae. These data support the development of OMC as a much needed option in the treatment of UTI caused by resistant Enterobacterales.Disclosures Tyler J. Stone, PharmD, Paratek (Research Grant or Support) Abdullah Kilic, MD, Paratek (Grant/Research Support) John Williamson, PharmD, Paratek (Research Grant or Support) Elizabeth Palavecino, MD, Paratek (Grant/Research Support)Paratek (Grant/Research Support)

Public Health Risk Assessment of the Door Handles of the Community Pharmacies in Qassim Region, Saudi Arabia

Door handles are being reported to harbor a diverse group of microorganisms, mainly bacteria. Presence of pathogenic and antibiotic-resistant bacteria in the door handles carry risk to the health of the public. For this reason, a study was carried in the Qassim region of Saudi Arabia by isolating bacteria from the pharmacy door handles from four different areas. Total 100 samples were collected by wiping the door handles with a sterile cotton swab soaked in sterile water. Microorganisms were isolated using Blood agar and MacConkey agar and identified following standard microbiological procedure. Siemens MicroScan Walkaway system was used for determination of antibiotic susceptibility pattern. In total, 301 bacteria from 13 bacterial species were isolated and identified. The predominant bacterial species include Staphylococcus spp. 56.48% followed by Bacillus spp. 12.29% and Micrococcus spp. 10.30%. Gram-negative bacteria like Shigella sonnei and Salmonella paratyphiA were also isolated. Being the most predominant species, Antibiotic resistance pattern of 39 Staphylococcus spp. were determined. 38.46% of the Staphylococcus spp. were found to be resistant to Cefoxitin, and 30.76% were beta-lactamase producing. The results also indicated that about one -third of Staphylococcus spp. were methicillin resistant. The door handles of pharmacies in the Qassim region carry risk to the health of the public. Proper hygienic measures are recommended for the public health safety until doors are made automatic and touch-free.