MALDI-TOF MS Research Articles

Immunoassay–mass spectrometry to identify Brucella melitensis

Two factors frequently impede accurate bacterial identification using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): inadequate bacterial abundance in real samples and bacterial combinations. For MALDI-TOF MS analysis and libraries for bacterial identification, time-consuming culture procedures are necessary to achieve sufficient concentration and isolation of a single bacterium. When dealing with hazardous bacteria like Brucella, which are more difficult to handle and cure, this problem becomes even more crucial. To overcome these obstacles, Fe3O4 magnetic nanoparticles (MNPs) linked with Brucella-specific antibodies and MALDI-TOF MS analysis have been used to create a quick and accurate technique for direct bacterial separation and identification in complex samples. This method allows MNPs to immune-selectively collect Brucella cells, which are then deactivated and ready for MALDI-TOF MS analysis by a formic acid/acetonitrile wash. Rabbits were used to manufacture brucella antibodies, which have effectively adsorbed onto the MNPs–protein A. Any particular Brucella bacteria found in the media might be absorbed by this MNPs–protein A–antibody immunoprobe. The concentration of Brucella bacterial cells increases the protein spectrum’s visibility by a factor of 103, making it possible to quickly identify Brucella spp. without first growing them in cultural conditions. This method has been successfully used to achieve a limit of detection (LOD) of 50 CFU/mL in an aqueous medium and genuine sample—milk. The diagnostic time for this harmful bacterium is greatly decreased because the entire procedure from bacterial isolation to species identification is finished in less than 60 min. High sensitivity and specificity are demonstrated by the immunoassay–MS approach, as the spectral pattern it produces matches well-known databases like SPECLUST and Ribopeaks.

Identification of Bed Bugs from Comoros, Using Morphological, Matrix-Assisted Laser Desorption Ionisation Time-of-Flight Mass Spectrometry, and Molecular Biology Tools, and the Detection of Associated Bacteria

After virtually disappearing from domestic dwellings in the Western world at the end of the Second World War, bed bugs have re-emerged in recent years. Few studies, however, have been carried out on these insects in tropical islands. In this study, we focussed on describing bed bug specimens collected from dwellings in a high-altitude village in Grande Comore, an island in the Comoros, in the Indian Ocean. We also aimed to detect the bacteria associated with them. Using MALDI-TOF MS coupled with molecular biology, we were able to confirm that the C. hemipterus species (the tropical bug) was the bug infesting these homes. Interestingly, the results also show that MALDI-TOF MS can differentiate between the developmental stages of bed bugs (immature and adult). Screening for bacteria was carried out using qPCR, regular PCR, and sequencing, with only Wolbachia DNA being found. Widespread surveys throughout the country are needed to ascertain the level of bed bug infestation, with a view to implementing appropriate control measures.

Salivary peptidomic profiling of chronic gingivostomatitis in cats by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and nanoscale liquid chromatography-tandem mass spectrometry.

Chronic gingivostomatitis in cats (FCGS) is a moderately to severely painful condition, potentially caused by inadequate immune response to oral antigenic stimulation. Salivary peptidome analysis can identify inflammatory protein mediators and pathways involved in oral mucosal immune activation and may indicate potential therapeutic options for FCGS. Evaluate the diversity and abundance of salivary peptides in cats with FCGS using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Thirty-two cats with FCGS and 18 healthy controls. Case-control cross-sectional study. We compared the salivary peptide profiles of diseased and healthy cats. The diagnosis of FCGS was confirmed by histopathology. Saliva samples were analyzed for viral infections using polymerase chain reaction (PCR), peptide mass fingerprint (PMF) using MALDI-TOF MS, and peptide identification using nano LC-MS/MS. Distinct clusters of peptide profiles were observed between groups. In FCGS, 26 salivary peptides were altered, including apolipoprotein A1, nuclear receptor subfamily 1 group I member 3, fibrinogen alpha chain, interleukin 2 receptor gamma, interleukin 23 receptor, hemoglobin subunit alpha, and serpin peptidase inhibitor clade A (alpha-1 antiproteinase, antitrypsin) member 12, protein-tyrosine-phosphatase, and cholinergic receptor nicotinic alpha 10 subunit. Protein-anti-inflammatory drug interaction networks were observed. Peptide mass fingerprint and peptide profiles identified distinct clusters between FCGS and healthy cats. The 9 novel salivary peptide markers were associated with the JAK/STAT and PI3K/Akt pathways and immune responses. These potentially noninvasive biomarkers may facilitate understanding of FCGS pathophysiology and guide future therapeutic research.

Size-dependent dynamics and tissue-specific distribution of nano-plastics in Danio rerio: Accumulation and depuration

Nano-plastics (NPs), defined as particles smaller than 1µm, have emerged as a significant environmental contaminant due to their potential ecological impacts. This study explores the size-dependent dynamics and tissue-specific distribution of polystyrene nano-plastics (PS-NPs) in Danio rerio exposed to PS-NPs at an environmentally relevant concentration of 1μg/mL for 28 days, followed by a 17-day depuration period. PS-NPs of 20, 100, 200, and 500nm were assessed in the intestine, liver, gills, muscle, and brain using transmission electron microscopy (TEM) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Smaller PS-NPs (20nm) showed the highest accumulation in the intestine, followed by the liver, and gills, due to their greater surface area and cellular penetration. In contrast, larger PS-NPs (500nm) exhibited lower accumulation and clearance rates, especially in the brain, suggesting restricted passage through biological barriers. The intestine consistently had the highest concentrations in both accumulation and depuration, while the brain maintained the lowest across all nanoparticle sizes. During depuration, smaller particles cleared more quickly, whereas larger particles persisted. This study highlights the tissue-specific distribution and retention patterns of PS-NPs in D. rerio, providing insights into nanoparticle behavior in aquatic organisms and the need for long-term size-specific environmental risk assessments.

A Comparative Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and Conventional Methods for the Diagnosis of Dermatophytes

A Comparative Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and Conventional Methods for the Diagnosis of Dermatophytes

Molecular Tracking of Emerging Fusarium Species in Keratitis: F. veterinarium, F. contaminatum, and F. curvatum.

Fungal keratitis, which is caused primarily by Neocosmospora and Fusarium species, is a significant global health issue that affects more than a million people annually in tropical and subtropical regions. Neocosmospora solani (formerly Fusarium solani) is a leading cause of corneal infections, along with members of the Fusarium oxysporum species complex (FOSC). This study provides new insights by reporting a series of ocular fusariosis cases caused by FOSC members and presenting molecular evidence linking specific haplotypes within FOSC to human infections. We describe three cases of Fusarium keratitis selected from a comprehensive review of clinicopathological data in our institution's archives. These cases were chosen for their distinctive clinical presentations and the involvement of less common Fusarium species. Two of these patients were diagnosed with keratitis and anterior endophthalmitis, and the third patient had a corneal ulcer previously treated with topical antivirals and antibiotics. All patients were successfully treated with topical amphotericin B. The Fusarium isolates from these patients were subjected to detailed molecular characterization, including DNA sequencing (tef1α, rpb2, CaM, tub2, and LSU), amplified fragment length polymorphism (AFLP) marker analysis, and MALDI-TOF MS analysis. Remarkably, our study reports the first case of human infection by F. veterinarium, alongside cases involving F. contaminatum and F. curvatum. Furthermore, a molecular survey using haplotypic networks based on tef1α sequences identified genotypes associated with human infections and revealed the emergence of F. veterinarium clade VII. Our findings emphasize the need for vigilance regarding emerging species within the FOSC, particularly F. veterinarium. This highlights the need for improved diagnostic tools and targeted research to combat fusarioid-related infections effectively.

A MALDI-ToF mass spectrometry database for identification and classification of highly pathogenic bacteria

Today, MALDI-ToF MS is an established technique to characterize and identify pathogenic bacteria. The technique is increasingly applied by clinical microbiological laboratories that use commercially available complete solutions, including spectra databases covering clinically relevant bacteria. Such databases are validated for clinical, or research applications, but are often less comprehensive concerning highly pathogenic bacteria (HPB). To improve MALDI-ToF MS diagnostics of HPB we initiated a program to develop protocols for reliable and MALDI-compatible microbial inactivation and to acquire mass spectra thereof many years ago. As a result of this project, databases covering HPB, closely related bacteria, and bacteria of clinical relevance have been made publicly available on platforms such as ZENODO. This publication in detail describes the most recent version of this database. The dataset contains a total of 11,055 spectra from altogether 1,601 microbial strains and 264 species and is primarily intended to improve the diagnosis of HPB. We hope that our MALDI-ToF MS data may also be a valuable resource for developing machine learning-based bacterial identification and classification methods.

Purification and characterization of lipase from Bacillus subtilis kubt4 for biodiesel production

The synthesis of biodiesel, a vital step in the search for sustainable energy sources, frequently entails costly procedures in which lipases are vital. This research focuses on the purification and characterization of lipase isolated from Bacillussubtiliskubt4, emphasizing its potential application in biodiesel production. The enzyme was purified using chromatographic techniques and characterized for its biochemical properties, substrate specificity, and stability under various conditions. Insights gained underscore its suitability as a biocatalyst for efficient biodiesel synthesis. To determine whether a lipase enzyme produced from Bacillus Subtilis kubt4 (Gene Bank Accession Number SUB3191429) has potential for use in industrial settings, this work focused on its purification and characterization. Three major chromatographic phases composed the enzyme purification procedure, which produced a lipase with a specific activity of 11.64 U/mg protein and a recovery yield of 11.27% after a 7.36-fold purification. SDS‒PAGE analysis revealed a molecular mass of 30 kDa, while MALDI-TOF MS and Mascot search analysis confirmed that the purified protein was a lipase. The characterized lipase exhibited alkaline properties, displaying optimal activity and stability at pH 8.0 and a temperature of 35 °C. The presence of Mg2+ ions significantly enhanced the enzyme activity by 180.21%. Kinetic analysis revealed Vmax and Km values for pNP palmitate of 8.33 M and 1.025 μM/min, respectively. Furthermore, the purified lipase was employed in the transesterification of Pongamia oil for biodiesel production. The biodiesel profile obtained from Pongamia oil demonstrated its suitability and sustainability as a biofuel source. Overall, this work clarifies the possible industrial use of purified lipase from Bacillus Subtilis kubt4 in the manufacturing of premium, eco-friendly biodiesel.

Identifying falsified COVID-19 vaccines by analysing vaccine vial label and excipient profiles using MALDI-ToF mass spectrometry

The rapid development and worldwide distribution of COVID-19 vaccines is a remarkable achievement of biomedical research and logistical implementation. However, these developments are associated with the risk of a surge of substandard and falsified (SF) vaccines, as illustrated by the 184 incidents with SF and diverted COVID-19 vaccines which have been reported during the pandemic in 48 countries, with a paucity of methods for their detection in supply chains. In this context, matrix-assisted laser desorption ionisation-time of flight (MALDI-ToF) mass spectrometry (MS) is globally available for fast and accurate analysis of bacteria in patient samples, offering a potentially accessible solution to identify SF vaccines. We analysed the COVISHIELD™ COVID-19 vaccine; falsified versions of which were found in India, Myanmar and Uganda. We demonstrate for the first time that analysis of spectra from the vaccine vial label and its adhesive could be used as a novel approach to detect falsified vaccines. Vials tested by this approach could be retained in the supply chain since it is non-invasive. We also assessed whether MALDI-ToF MS could be used to distinguish the COVISHIELD™ vaccine from surrogates of falsified vaccines and the effect of temperature on vaccine stability. Both polysorbate 80 and L-histidine excipients of the genuine vaccine could be detected by the presence of a unique combination of MALDI-ToF MS peaks which allowed us to distinguish between the genuine vaccines and falsified vaccine surrogates. Furthermore, even if a falsified product contained polysorbate 80 at the same concentration as used in the genuine vaccine, the characteristic spectral profile of polysorbate 80 used in genuine products is a reliable internal marker for vaccine authenticity. Our findings demonstrate that MALDI-ToF MS analysis of extracts from vial labels and the vaccine excipients themselves can be used independently to detect falsified vaccines. This approach has the potential to be integrated into the national regulatory standards and WHO’s Prevent, Detect, and Respond strategy as a novel effective tool for detecting falsified vaccines.

Quantitative MALDI-TOF Mass Spectrometry of Star-Shaped Polylactides Based on Chromatographic Hyphenation.

The end groups of three- and four-arm star-shaped polylactides (PLA) with trimethylolpropane and pentaerythritol core structures were functionalized with acetic acid. Reaction products with different degrees of functionalization were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Additional gradient elution liquid adsorption chromatography (GELAC) measurements were performed to determine the degree of functionalization. This technique enabled clear separation and sufficient quantification of the formed species. These chromatographic data could be used inversely to quantify mass spectrometric results, which are usually biased by the unknown ionization probabilities of different polymer end group structures. Our results showed that, in this particular case, the peak intensity in the MALDI-TOF mass spectra can be used to semiquantitatively determine the degree of functionalization in incompletely functionalized multiarm PLA.

P-1588. Effect of Multiplex Polymerase Chain Reaction (PCR) versus Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MF) on Time to Optimal Therapy in Patients with Positive Blood Cultures

Abstract Background Rapid diagnostic technology is an important tool that can be used to optimize antimicrobial therapy for patients with bloodstream infections Several technologies for rapid organism identification exist including matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and multiplex polymerase chain reaction (PCR). The primary objective is to compare time to optimal antimicrobial therapy with MALDI-TOF MS vs multiplex PCR rapid diagnostic systems for patients with positive blood cultures. Methods This multicenter, retrospective, cohort study included patients with a positive blood culture and identification via MALDI-TOF MS (June 1st – June 7th, 2021) compared to multiplex PCR (June 1st – June 7th, 2022). Patients were excluded if expired or discharged before organism identification, transferred from an outside hospital with a positive blood culture, if rapid diagnostics not performed, if identified pathogen had no defined optimal therapy per system guidelines or polymicrobial infection. The primary outcome was time to optimal therapy (TTOT). Secondary outcomes included time to effective therapy (TTET), length of stay, and clinical failure defined as recurrence of bacteremia within 30 days or in-hospital mortality. Results A total of 221 patients were included (MALDI-TOF MS = 117; multiplex PCR = 104). Utilization of multiplex PCR compared to MALDI-TOF MS significantly reduced overall TTOT (21.1 hours vs 41.1 hours, p < 0.01). This significant reduction in TTOT with multiplex PCR was observed with gram-negative bacteremia (4 hours vs 37 hours, p < 0.01) and gram-positive bacteremia (17.8 hours vs 41.1 hours, p < 0.01) but was not statistically significant for blood cultures deemed contaminants (42 hours vs 43.5 hours, p=0.58). TTET was improved for multiplex PCR although not statistically significant (1.5 hours vs 7.5 hours, p=0.06). No differences were observed for clinical failure or hospital length of stay. Conclusion Multiplex PCR significantly decreased time to optimal antimicrobial therapy when compared to MALDI-TOF MS for patients with positive blood cultures. Disclosures All Authors: No reported disclosures

Investigation of Factors Associated With Bovine Mastitis in a Compost Barn System and Identification of Microorganisms Involved, Using Maldi-Tof Mass Spectrometry

Objective: To investigate factors associated with bovine mastitis in a Compost Barn system and to use MALDI-TOF mass spectrometry to determine the microorganisms involved in the infection. Theoretical Reference: Mastitis negatively impacts dairy farming. To target control measures efficiently, it is important to assess predisposing factors and identify microorganisms. The MALDI-TOF MS technique can contribute to this. Method: Subclinical mastitis was diagnosed by the California Mastitis Test (CMT), with milk collected from positive teats for microbiological analysis by Gram staining and subsequent identification by MALDI-TOF mass spectrometry. Teats were classified for hyperkeratosis and dirt according to standardized methodologies. The seasonal influence on mastitis was analyzed based on local climate data. Results and Discussion: The prevalence of mastitis in the Compost Barn was 35%, with a higher frequency in the warmer, rainier months. There was an association between the occurrence of mastitis and hyperkeratosis (p<0.000001) and dirt (p = 0.02) in the Compost Barn system. Staphylococcus and Bacillus predominated in cases of mastitis. There was no dependence between the type of microorganism and the degree of subclinical mastitis (p = 0.053). Research implications: The results may contribute to developing efficient strategies to combat and control bovine mastitis in the dairy sector. Originality/value: This study contributes to the literature by showing the association of factors with the occurrence of bovine mastitis on dairy farms in the north of Minas Gerais. Using the MALDI-TOF MS technique, it also contributes to the libraries of microorganisms involved in cases of mastitis. The relevance and value of this research are evidenced by its influence on the decision-making process of dairy farmers.

P-1023. Prevalence and distribution of fungal microorganisms among cutaneous specimens submitted to community microbiology laboratories

Abstract Background Not much is known about the local prevalence of cutaneous fungal infections in Canada because these data are not readily available. Knowing the distribution of microorganisms in local settings may help developing guidance for empiric therapy, especially for immunocompromised patients whose cutaneous fungal infections could turn into systemic infections. The current retrospective study aimed to establish the prevalence and distribution of fungal microorganisms among patients who had specimens submitted to community microbiology laboratory for cutaneous fungal culture workups. Methods LifeLabs regional microbiology laboratories, connected with 129 collection centers in urban and rural communities in British Columbia, provided reports of isolated fungus from hair, nail, and skin scraping specimens from 1 January 2022 to 31 December 2022. Culture followed by microscopy and/or MALDI-TOF mass spectrometry was routinely used to isolate and identity microorganisms, but nucleic-acid testing was added to help identification if needed. Results Among the 20652 cutaneous specimens submitted in a year, 3665 of them were reported to have microorganism growth. The most reported microorganism was Trichophyton species (43.8%), followed by Penicillium species (5.8%), Candida albicans (4.2%), Aspergillus species (3.6%), and Fusarium species (2.8%). Of note, other than Trichophyton species, the other two main dermatophytes Microsporum species and Epidermophyton species accounted for only 0.2% and 0.1% of the positive specimens, respectively. Pseudomonas aeruginosa was incidentally identified in 2.9% of the positive specimens in the fungal culture workups. Conclusion Trichophyton species was the most reported microorganism among the hair, nail, and skin scrapping specimens submitted for cutaneous fungal culture workups from communities in British Columbia. Pseudomonas infections, which could present as chloronychia, folliculitis, ecthyma gangrenosum, could be considered in the differential diagnoses of cutaneous fungal infections. The current data will help clinicians to determine the empiric therapy needed based on each patient’s clinical situation. Disclosures All Authors: No reported disclosures

P-352. Resistance of the Public Health Threat Emerging Pathogen Candida Auris from Multiple Sources to Antifungal Agents (ARIA 2020-2022)

Abstract Background As highlighted by the Centers for Disease Control (CDC), resistance to antifungal therapies leads to high morbidity and moderate to severely high mortality. In recent years, infections caused by Candida auris have been extremely difficult to treat and C. auris is now labelled as a public health threat. ARIA is a global surveillance initiative collecting yeast and fungal isolates from worldwide sources designed to determine resistance to antifungal agents and trends over time. The current study reports C. auris isolates from the ARIA project and outlines the susceptibility of these isolates to clinically used antifungal agents. Methods A total of 62 clinical isolates of C. auris were collected between 2020 and 2022. These originated from specific hospital sites in Germany (1), India (26), Kuwait (5), Panama (20) and the United Arab Emirates (10). All isolates were non-consecutive non-duplicate isolates. Isolates were identified via MALDI-TOF mass spectroscopy and susceptibility testing was performed in line with Clinical Laboratory Standard Institute (CLSI) guidelines using amphotericin B, anidulafungin, caspofungin, fluconazole, isavuconazole, micafungin, posaconazole and voriconazole. As there are no accepted breakpoints for C. auris, CDC tentative e breakpoints were adopted. Results MIC distributions with respect to CDC tentative breakpoints specific for C. auris are shown in the Table. 14 of the 62 isolates (23%) were resistant to amphotericin B, 1 isolate (1.6%) resistant to caspofungin, 53 isolates (85%) resistant to fluconazole and 1 isolate resistant to micafungin. Notably, all amphotericin-B resistant isolates were also fluconazole-resistant. Conclusion The data from the ARIA study further emphasize the threat posed by C. auris considering its ability to resist commonly used antifungal agents. Continued monitoring of C. auris susceptibility is essential. Disclosures All Authors: No reported disclosures

100. Genomic epidemiology of carbapenemase-producing Enterobacter species in Toronto, Canada, 2007-2021

Abstract Background Identification of transmission networks of carbapenemase-producing organisms (CPO) is critical to identifying their reservoirs and limiting their spread. This study aimed to use whole-genome sequencing to identify genomic relationships between CP Enterobacter (CP-Ent) in Ontario, Canada over a 15-year period. Methods All CP-Ent cases identified by prospective population-based surveillance in the Toronto/Peel Region from first isolate in 2007 to 2021, from hospital sinks (2016-2019), and wastewater treatment plants and surface water sites (2015-2017) were included. CPO isolates were identified by phenotypic screening (ertapenem MIC > 1mg/L or meropenem disc diffusion diameter ≤ 25mm), confirmed as Enterobacter by MALDI-TOF MS and as positive for carbapenemase genes by PCR, and sequenced by Illumina. Genomics analysis utilized a custom pipeline combining Snippy, IQ-Tree, and ClonalFrameML. Results Overall, 182 isolates from 127 patients, 61 isolates from 40 sinks, and 155 isolates from 9 wastewater plants/surface water sites were available (Table 1). Thirty-one of 127 (24%) patients had > 1 sequenced isolate: in 29 patients all isolates were highly related (median SNV distance 3.5, range 0-13); one patient had two CP-Ent species ∼1.5 years apart, and one had 2 sequence types (STs) 86 days apart. Of 12 (30%) sinks with > 1 isolate, 5 had 2 STs recovered at different times (4 with different carbapenemases). Multiple species, STs, and carbapenemases were recovered from sewage trunks and surface water. Using a ≤ 20 SNV threshold between any one pair of isolates, 14 multi-patient putative clusters involving 39 patients were identified; 1 added cluster was identified and 4 expanded when the threshold was increased to 40 SNVs (Table 2). At the 40 SNV threshold, the median number of patients per cluster was 2 (range 2-9), and the median number of hospitals with at least one first identified patient in each cluster was 1.5 (range 1-4). In total, 35% (45/127) patients were part of a cluster. Conclusion CP-Ent in Ontario are diverse, but a significant minority of affected patients are part of genomically-defined clusters. Genomic clusters may reflect undetected transmission of CP-Ent in healthcare, exposure to water, or other as yet unidentified sources. Disclosures Allison McGeer, MD, AstraZeneca: Honoraria|GSK: Honoraria|Merck: Honoraria|Moderna: Honoraria|Novavax: Honoraria|Pfizer: Grant/Research Support|Pfizer: Honoraria|Roche: Honoraria|Seqirus: Grant/Research Support|Seqirus: Honoraria

A novel combinatorial approach for the Identification of Cutibacterium namnetense and Cutibacterium modestum from Facial Acne Samples

Background: Cutibacterium spp. is one of the most understudied bacteria and this is owed to its slow growing nature and its stringent requirement for anoxic conditions. To date, shortgun metagenomic sequencing and MALDI-TOF MS are widely used for species detection but, the latter is not able to distinguish C. acnes from C. modestum and C. namnetense. Our study has innovatively combined colony morphology, biochemical assays and16s rRNA gene sequencing to identify C. acnes as well as the underreported C. namnetense and C. modestum from facial clinical acne samples.Methods: The clinical samples were obtained using a non-invasive method from acne patients at the Dermatology Clinic of Hospital Tuanku Jaafar, Seremban, Malaysia between January 2022 to December 2022. Colonies of Cutibacterium spp. were screened on BHI agar followed by subjecting them to the catalase and indole tests. The isolates were verified as Cutibacterium spp. using API20A and 16s rRNA Sanger gene sequencing.Result: Out of 68 Cutibacterium spp. isolates, 3 were identified as C. modestum and 1 as C. namnetense while the rest were C. acnes. All isolates were present as raised, white colonies with 0.03 to 1mm in diameter on BHI agar. 89.71% of these isolates were indole producers. All isolates were identified as C. acnes in API20A but, the 16srRNA gene sequencing revealed 4 isolates as C. modestum and C. namnetense.Conclusion: This study is the first to report the isolation of C. namnetense and C. modestum in clinical facial acne samples from Malaysia and across Asia, employing a modified combination of morphological, biochemical, and 16srRNA gene analyses. This methodical yet straightforward approach serves as a viable alternative in research settings lacking access to advanced techniques like MALDI-TOF and shotgun metagenomic sequencing. Moreover, this conventional isolation approach is valuable in assessing the sensitivity of the isolates to inhibitory agents apart from antibiotics, expanding researchers' abilities to develop potent antibacterial agents required for human health and wellbeing.Keywords: Acne; Clinical Samples; Combinatorial; Identification

P-2163. Inconsistent identification of Corynebacterium species by MALDI-TOF MS and molecular characteristics of Corynebacterium jeikeium complex isolates causing invasive infections in Japan

Abstract Background Corynebacterium spp. is increasingly identified due to widespread use of MALDI-TOF MS in clinical laboratories. However, accuracy of identification of Corynebacterium spp. and genetic characteristics of clinical isolates are unclear. Methods We collected 17 isolates from cases diagnosed as infections caused by Corynebacterium spp. at a single center in Japan. Species identification was performed using MALDI Biotyper with reference library BDAL v11 (Bruker). Whole-genome sequencing (WGS) was also performed using MiSeq (Illumina), and the species were genetically identified using a threshold of ≥ 95% average nucleotide identity (ANI) with type strains. Regarding Corynebacterium jeikeium complex, an additional analysis was performed using 32 isolates detected from multiple sets of blood cultures at eight additional centers in Japan.Figure.Phylogenetic tree of Corynebacterium macclintockiae clinical isolates collected in this study.Pairs of strains detected at the same facility with SNP differences of < 20/genome per year difference in isolation time are marked with red squares. Results In the single-center study, eight isolates (2: blood, 5: intraabdominal fluid, 1: urine) and three isolates (3: intraabdominal fluid) were identified as Corynebacterium striatum and Corynebacterium amycolatum, respectively, by MALDI Biotyper, and the identification was confirmed by WGS. However, all six isolates identified as C. jeikeium by MALDI Biotyper (1: blood, 4: intraabdominal fluid, 1 perivertebral abscess) had an ANI of 87-88% with C. jeikeium NCTC 11915T and were genetically identified as Corynebacterium macclintockiae with ANI of 97-98% with C. macclintockiae c9Ua_112T (Table). Of the 32 isolates detected in blood cultures at eight institutions and identified as C. jeikeium by MALDI Biotyper, 31 were identified as C. macclintockiae by WGS (Table). Core-genome phylogenetic analysis of 37 C. macclintockiae isolates identified multiple clades spread in the same center and three pairs of isolates with SNP differences of < 20/genome per year difference in isolation time (Figure). Conclusion Identification of C. striatum and C. amycolatum by MALDI Biotyper was consistent with the genetic identification. The vast majority of C. jeikeium complex isolates causing infections in Japan were genetically identified as C. macclintockiae, but MALDI Biotyper identified them as C. jeikeium with high score values. SNP analysis suggested that some C. macclintockiae isolates may spread by nosocomial transmission. Disclosures Sohei Harada, MD, PhD, MSD: Honoraria|Shionogi: Honoraria Tomoo Saga, M.D., Ph.D., Asahi Kasei Pharma: Honoraria|Beckman-Coulter: Honoraria|Miyarisan Pharmaceutical: Honoraria|Pfizer: Honoraria|Shionogi: Honoraria|Sumitomo Pharma: Honoraria Yohei Doi, MD, PhD, bioMerieux: Lecture fees|Entasis: Grant/Research Support|Fujifilm: Advisor/Consultant|Gilead Sciences: Advisor/Consultant|GSK: Advisor/Consultant|KANTO CHEMICAL CO.,INC.: Grant/Research Support|KANTO CHEMICAL CO.,INC.: Patent for genotyping kit|MeijiSeika Pharma: Advisor/Consultant|Moderna: Advisor/Consultant|MSD: Lecture fees|Pfizer: Advisor/Consultant|Shionogi & Co., Ltd.: Grant/Research Support|Shionogi & Co., Ltd.: Lecture fees Kyoko Yokota, M.D, MSc, Chugai pharmaceutical Co.,Ltd: Honoraria|Meiji Seika Pharma Co.,Ltd: Honoraria Jun Suzuki, M.D., Eiken Chemical: Honoraria|Kyorin Pharmaceutical: Honoraria Koki Kikuchi, M.D., bioMérieux: Honoraria|Pfizer: Honoraria Kazuhiro Tateda, PHD, GSK: Honoraria|Moderna: Honoraria|Pfizer: Honoraria|Shionogi: Honoraria

P-1096. Activity of Ibrexafungerp and Comparator Antifungals Tested against Candida and Aspergillus Isolates Collected from Invasive Infections in a Global Surveillance Program in 2023

Abstract Background Ibrexafungerp (IBX) is a novel triterpenoid antifungal agent that was approved by the U.S. FDA for the treatment of vulvovaginal candidiasis (VVC) in 2021. Further studies are ongoing to assess the efficacy of IBX for the treatment of invasive candidiasis (IC) and other refractory fungal infections, including aspergillosis. CLSI, EUCAST, or FDA clinical breakpoints or epidemiological cutoff values are not yet defined for IBX against fungi. We evaluated the activity of IBX and comparator agents against clinical isolates from IC and invasive aspergillosis infections collected from a global surveillance program in 2023. MIC50/90 (mg/L) for ibrexafungerp (IBX) and comparator agents against 5 most common Candida spp., C. auris, and 3 most common Aspergillus spp. in 2023 SENTRY Antifungal Surveillance Program Methods A total of 1120 Candida spp. and 204 Aspergillus spp. isolates consecutively collected as part of the SENTRY Program were evaluated. Isolates were recovered from 11 medical centers in North America, 18 in Europe, 7 in Asia-Pacific, and 5 in Latin America. Identification was performed by MALDI-TOF MS and/or sequencing and susceptibility testing was performed using the CLSI broth microdilution method for IBX, anidulafungin (AND), micafungin (MFG), fluconazole (FLC), itraconazole (ITC), and voriconazole (VRC). Results Table 1 displays MIC50/90 results for the 5 most common Candida spp., C. auris, and the 3 most common Aspergillus spp. The IBX MIC50/90 results were low against all organisms including organisms of critical concern like C. auris. Among Candida species, IBX had similar or higher MIC50/90 to AND except for C. parapsilosis where IBX MIC50/90 values were 4- to 8-fold lower. MFG was more potent (4- to 16-fold) against most Candida spp. except for C. parapsilosis where IBX had 2-to 4- fold lower MIC50/90 and C. auris where IBX was similar to MFG. Notably, the highest MIC result observed for IBX against C. auris was 1 mg/L. MIC50/90 values for IBX were also 8- to > 256-fold lower than azoles against Aspergillus spp. but higher (2- to ≥ 16-fold) than MFG or AND. Conclusion IBX has good activity against the predominant Candida and Aspergillus species from a worldwide contemporary collection. This agent may represent an oral option for the management of difficult-to-treat invasive fungal pathogens such as C. glabrata, C. auris, and Aspergillus spp. from diverse infection sources in addition to its current indication for VVC. Disclosures Marisa Winkler, MD, PhD, Element Iowa City (JMI Laboratories) was contracted to perform services in 2023 for > 30 biotech and pharmaceutical companies: Grant/Research Support Sharon Min, MS, GSK: Employee

Complete genome sequence of an Enterococcus devriesei strain isolated from Zophobas morio larvae: Complete genome of Enterococcus devriesei Ed-CK-24.

.Enterococcus spp. are typically found in multiple settings and are sometimes responsible for difficult-to-treat infections. In this context, very little is known about E. devriesei, a rare species first isolated from a river lamprey. Importantly, no complete genome of E. devriesei currently exists in public repositories. . An E. devriesei strain (Ed-CK-24) was isolated from homogenized Zophobas morio larvae. Initial species identification (ID) was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing (AST) was performed by broth microdilution. Whole-genome sequencing (WGS) was done using both Illumina NovaSeq 6000 and Nanopore MinION to generate a complete genome assembly. WGS data were used to confirm species ID, antimicrobial resistance genes (ARGs) and plasmid replicon sequences screening. A database search for other E. devriesei genomes in NCBI was performed and used for 16S rRNA and core-genome phylogeny analyses. . WGS and bioinformatic analyses were performed, resulting in a complete genome assembly and allowing accurate taxonomic classification of Ed-CK-24 strain as E. devriesei. Alignment of 16S rRNA sequences of representative Enterococcus spp. further supported the ID of Ed-CK-24. A core-genome phylogenetic analysis revealed no clonal relationships between Ed-CK-24 and other E. devriesei derived from multiple sources. The Ed-CK-24 strain was resistant to clindamycin and quinupristin/dalfopristin. ARG screening identified the lsa(A) gene, carried on a 633,497-bp circular megaplasmid. . WGS of Ed-CK-24 allowed high-resolution genomic comparison and epidemiologic analysis of E. devriesei. Its isolation from Z. morio larvae, commonly used in pet food, warns for further surveillance as the human pathogenic potential of this species remains unknown.

P-1095. Activity of Rezafungin against Clinical Isolates of Uncommon Species of Candida spp

Abstract Background Rezafungin (RZF) is a long-acting echinocandin recently approved by the FDA to treat invasive candidiasis (IC) and candidemia. RZF CLSI breakpoints are available for Candida albicans, C. auris (CARS), C. dubliniensis (CDUB), C. glabrata, C. krusei, C. parapsilosis (CPRP), and C. tropicalis. However, RZF susceptibility results are scarce against other Candida spp. We evaluated in vitro activity of RZF, anidulafungin (AND), caspofungin (CAS), micafungin (MCF), fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB) against less common species of Candida spp. including CARS and CDUB. Methods We tested 590 (1/patient) less common Candida spp. isolates from IC infections as part of the SENTRY Program (Table 1). Isolates were collected from 2020-2022 in 80 hospitals including 31 in North America, 29 in Europe, 14 in Asia-Pacific, and 6 in Latin America. Isolates were identified by MALDI-TOF MS and/or sequencing and susceptibility testing was performed using the CLSI broth microdilution method. CLSI breakpoints or epidemiologic cutoff values were applied where available. Results RZF displayed similar activity to other echinocandins (within ±2-fold for MIC50/90 values) against all Candida spp. tested (Table 2). The lowest RZF MIC50/90 values were noted against C. kefyr (n=51, 0.03/0.06 mg/L) and C. pelliculosa (n=10, 0.015/0.03 mg/L). Higher RZF MIC50/90 values were noted for C. guilliermondii (CGU, n=33, 1/1 mg/L) and for isolates in the CPRP complex (C. orthopsilosis (CORT) n=49, 0.5/1 mg/L, C. metapsilosis (CMET) n=25, 0.12/0.5 mg/L). The same pattern was seen with other echinocandins against these Candida spp. RZF was active against 97.7% of CDUB and 95.4% of CARS isolates applying the CLSI breakpoints. There were three CARS isolates that were resistant to RZF with MICs of 1- >4 mg/L. Two of these were susceptible to CAS and one was resistant or non-wildtype to all echinocandins. For FLC 69.7% of CGU, 85.7% of CORT, 100% CMET, and only 10.8% of CARS isolates were susceptible. Conclusion RZF has potent in vitro activity against a large collection of less common Candida spp. as demonstrated by worldwide surveillance testing. The activity of RZF against these isolates was comparable to other echinocandins and generally better than FLC. Disclosures Marisa Winkler, MD, PhD, Element Iowa City (JMI Laboratories) was contracted to perform services in 2023 for > 30 biotech and pharmaceutical companies: Grant/Research Support