Candida spp. are important pathogens among intensive care unit (ICU) patients, and colonization is the first step toward invasive infection. This study describes the epidemiology, risk factors, and colonization dynamics of Candida in ICU patients.Figure 1.Days of therapy and days of antibiotic spectrum coverage among Candida spp. colonized and non-colonized patients. This nested case–control study included adult ICU patients who underwent fecal sample collection between 2021 and 2024. Candida colonization was detected via stool culture on Candida selective media and species were identified by MALDI-TOF. Patients were classified as either colonized or never colonized during ICU stay. Baseline characteristics, Total Days of Therapy (DOT), Days of Antibiotic Spectrum Coverage (DASC) and in-hospital mortality were compared. A time-dependent conditional logistic regression with a 1:2 matched design was used to identify risk factors for colonization, considering only exposures before the colonization day in cases and the matched day in controls.Figure 2.Temporal distribution of Candida species among ICU-colonized patients. Among 191 ICU patients, 108 (56.6%) were colonized by Candida spp. Colonized patients had higher rates of liver disease and diabetes (Table 1). Colonized patients had greater exposure to vancomycin, meropenem, and ceftriaxone, along with higher DOT and DASC (Figure 1). In colonized patients, C. albicans was the most common species (52%), followed by C. glabrata (43%) (Figure 2). Co-colonization with other Candida species occurred in 14% of patients, and 25% experienced spontaneous clearance during their ICU stay. Colonized patients also had higher Enterococcus spp. colonization (24.1%). Univariate analysis showed that prior exposure to meropenem (odds ratio [OR] 2.2, 95% confidence interval [CI] 1.4-3.4) and ceftriaxone (OR 2.5, CI 1.5-4.1) were risk factors for colonization, while DOT and DASC were not significantly different between groups (Table 2). Candidemia occurred exclusively in colonized patients, although in cases (n=5) bloodstream species differed from those found in stool. Mortality did not differ by colonization status. Candida spp. colonization was common among ICU patients and often involved multiple species. Colonization was linked to increased broad antibiotic exposure. Colonized patients were more likely to have candidemia, although infecting species differed from the colonizing isolates. All Authors: No reported disclosures

Candida auris is a drug-resistant fungus that poses a growing risk in healthcare environments, particularly among intensive care unit (ICU) patients. While other Candida species routinely colonize the gut, it is unknown if the gastrointestinal (GI) tract is a niche for C. auris. We aimed to determine the presence of C. auris in the gut of ICU patients and evaluate whether these isolates exhibit phenotypic traits that support intestinal colonization.Figure 1.Abbreviations: DCA, deoxycholic acid; ICU, intensive care unit; MALDI-ToF, matrix-assisted laser desorption/ionization-time of flight; WGS, whole genome sequencing.Figure 2.C. auris gut colonization and clinical detection in intensive care unit (ICU) patients. We assessed C. auris gut colonization in ICU patients by collecting stool samples and demographic/clinical information from enrolled patients from 01/2021-06/2024 (Figure 1). Stool samples were cultured on Candida selective media, isolates were identified using MALDI-ToF and their genomes were sequenced. To evaluate bile acid resistance, representative stool isolates of C. auris and C. albicans were exposed to increasing concentrations of the secondary bile acid deoxycholic acid (DCA), and growth was measured over time. Antifungal minimum inhibitory concentrations (MICs) were determined by broth microdilution.Figure 3.Phylogenetic tree of C. auris isolates (Clade III), collected in triplicate from each patient on different days from stool and clinical cultures.Figure 4.Growth of C. auris vs. C. albicans gut colonizing isolates in deoxycholic acid (DCA). Of 150 ICU patients enrolled, the mean age was 60.8 ± 15.6 years, 83 (56%) were male, and 24% were Hispanic/Latino. Mean Charlson Comorbidity Index was 4.9 ± 3.0; 34 patients (23%) were solid organ transplant recipients, and 54 (36%) were in shock on ICU admission. C. auris was detected in stool cultures of three patients (2%), of which two had colonization at other body sites (Figure 2). Genomic analysis showed that these isolates were highly clonal and belonged to Clade III (Figure 3). All C. auris isolates were resistant to fluconazole and susceptible to micafungin. When exposed to DCA, C. auris stool isolates exhibited significantly less growth inhibition compared to C. albicans stool isolates. At high (0.3%) DCA concentrations, the mean inhibition rates were 17% for C. auris stool isolates and 66% for C. albicans stool isolates (Figure 4). These findings provide evidence of C. auris GI colonization in ICU patients. Moreover, the demonstrated resistance of C. auris stool isolates to DCA highlights its potential adaptive mechanisms for survival in the gut. These findings indicate that the GI tract may be a persistent reservoir for C. auris with implications for transmission and infection control. All Authors: No reported disclosures

Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. ( C. cuniculus , C. hominis , C. meleagridis , C. parvum , C. tyzzeri , and C. viatorum ) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.

Pseudomonas aeruginosa is an opportunistic human pathogen and a frequent cause of multidrug-resistant infections. This organism continues to evade antimicrobial therapy despite the clinical introduction of new antipseudomonal antibiotics over the past several years. One of these agents is cefiderocol (FDC), a novel siderophore-cephalosporin conjugate antibiotic that was designed to overcome both intrinsic and acquired β-lactam resistance mechanisms in P. aeruginosa. However, studies have demonstrated that inactivation of energy transducer protein (TonB)-dependent receptors, most notably the catechol siderophore receptor piuA, can substantially curtail the drug's ability to permeate the bacterial outer membrane, leading to rapid development of resistance. In this study, we examined the FDC resistance mechanisms of the laboratory strain PA14. We demonstrated that inactivation of the ferripyochelin receptor FptA was a first-step mutation toward FDC resistance. Through transposon mutagenesis, we identified several resistance pathways following fptA inactivation, such as the loss of an additional FDC receptor and overexpression of the MuxABC-OpmB multidrug efflux system. Introduction of clinically identified mutations analogous to these transposon insertions in the absence of fptA conferred full FDC non-susceptibility while preserving the activity of other antipseudomonal β-lactam antibiotics. We also demonstrated that inactivation of fptA in a pyoverdine biosynthetic mutant disrupted bacterial iron homeostasis and conferred a fitness disadvantage. These FDC resistance mechanisms identified in PA14 highlight the long-term challenges of using FDC treatment for drug-resistant P. aeruginosa infections.

BACKGROUND:Cefiderocol retains high rates of activity against carbapenem-resistant P. aeruginosa (CRPA) and is being introduced as a last resort antibiotic in developing countries. This study assessed the clinical and genotypic characteristics associated with decreased cefiderocol susceptibility among a global collection of CRPA isolates. METHODS:Clinical isolates (n=186) selected from 972 patients enrolled from 10 countries as part of the Prospective Observational Pseudomonas (POP) study were characterized by whole-genome sequencing, cefiderocol broth microdilution, and population analysis profile. We compared clinical and genomic characteristics between susceptible isolates and those with reduced cefiderocol susceptibility (defined as heteroresistant by population analysis, or intermediate or resistant using current breakpoints). A hollow fiber infection model was used to evaluate the emergence of resistance under human serum simulated cefiderocol exposures. RESULTS:Of the 186 isolates, 11 (5.9%) were cefiderocol non-susceptible and 28 (15.1%) were heteroresistant using CLSI breakpoints. With EUCAST breakpoints, 20 (10.8%) were cefiderocol resistant and 20 (10.8%) were heteroresistant. Reduced susceptibility isolates were present across all geographic regions and harbored changes in PirRS-regulated siderophore receptors, AmpC β-lactamase mutations associated with decreased ceftolozane/tazobactam susceptibility, cpxS mutations, and the β-lactamases blaNDM and blaVEB. Resistance to ceftolozane/tazobactam and ceftazidime/avibactam were independently associated with reduced cefiderocol susceptibility. Cefiderocol resistance emerged in a heteroresistant, but not susceptible isolate at human simulated exposures. CONCLUSIONS:Decreased cefiderocol susceptibility is present worldwide and is not restricted to a clonal high-risk lineage. This raises concerns about the use of cefiderocol as a salvage regimen in patients with multidrug-resistant P. aeruginosa infection.

ABSTRACTTicks are obligate blood-feeding parasites associated with a huge diversity of diseases globally. The hard tickIxodes ricinusis the key vector of Lyme borreliosis and tick-borne encephalitis in Western Eurasia.Ixodesticks have large and repetitive genomes that are not yet well characterized. Here we generate two high-qualityI.ricinusgenome assemblies, with haploid genome sizes of approximately 2.15 Gbp. We find transposable elements comprise at least 69% of the twoI. ricinusgenomes, amongst the highest proportions found in animals. The transposable elements in ticks are highly diverse and novel, so we constructed a repeat library for ticks using ourI.ricinusgenomes and the genome ofI.scapularis, another major tick vector of Lyme borreliosis. To understand the impact of transposable elements on tick genomes we compared their accumulation in the twoIxodessister species. We find transposable elements in these two species to be drivers of genome evolution in ticks. TheI.ricinusgenome assemblies and our tick repeat library will be valuable resources for biological insights into this important ectoparasite. Our findings highlight that further research into the impact of transposable elements on the genomes of blood-feeding parasites is required.

Abstract Background Penicillin (PEN) and ampicillin (AMP) resistance is rare in E. faecalis, and may arise via increased expression of penicillin-binding protein 4 (PBP4), mutations affecting PBP4’s affinity to β-lactams, and rarely β-lactamase production. We recently identified 23 vanA vancomycin-resistant E. faecalis (VREfs), recovered from 19 patients in 4 hospitals in Chile (2020-2023) that exhibited PEN resistance and unusually elevated AMP MICs (4-16 µg/mL). Here, we characterize the molecular basis of the reduced β-lactam susceptibility in these VREfs isolates.Table 1.AMP susceptibility after inducible expression of pbp4SCL10298 in the pbp4-defective and AMP hypersusceptible strain JH2-2Δpbp4 Methods We analyzed 23 VREfs including sequence type (ST), resistome, PBP sequences and pbp4 promoter. PBP4 production was assessed by ELISA and Western blot using goat anti-rPBP4 sera, in the Chilean VREfs isolate SCL10298 and using pbp4-defective strains (JH2-2Δpbp4 and OG117Δpbp4). We recreated the pbp4 changes of SCL10298 by allelic replacement of E. faecalis OG117 (OG117 Δpbp4SCL10298) using CRISPR-Cas9. Expression of pbp4SCL10298 was also assessed in-trans in a JH2-2 background (JH2-2Δpbp4) using a nisin-inducing vector (pMSP3535).Figure 1.Schematic representation of predicted open reading frames (ORFs) on the JH2-2 and SCL10298 Black line represents the nucleotide sequence for the PBP4 promoter region, followed by the predicted ORFs indicated with grey arrows. Text in red represents the amino acid sequence sharing 100% identity between JH2-2 ORF1 and SCL10298 ORF1-p1 and ORF1-p2. Results All 23 isolates belonged to ST1518 (an ST6 single-locus variant). No β-lactamase-encoding genes or changes in pbppromoter regions were detected. A frameshift deletion of 100 nt was observed in pbp4 in all isolates, resulting in 2 open reading frames, including a potential transcript of a shorter pbp4 (ORF1-p1, fig1). All the other PBP sequences were identical to those of E. faecalis JH2-2. PBP4 was detected in SCL10298 by whole-cell ELISA. Further, western blots revealed no differences in PBP4 amounts between SCL10298 and control strains. Allelic replacement of OG117Δ did not result in decreased AMP susceptibility as compared to the parental OG117 strain (AMP MIC 0.5 µg/mL) nor did the inducible expression of the truncated version of pbp4 in JH2-2Δpbp4 (0.19 µg/ml, table1). Conclusion We document the emergence of a new lineage of VREfs in Chile with alteration in PBP4 that exhibits decreased susceptibility to B-lactams (akin E. faecium). Our molecular experiments suggest that an alteration of PBP4 is necessary, but not sufficient to increase the MICs of AMP. Therefore, we propose the strain background might be key for the full expression of the B-lactam-resistant phenotype. Disclosures William R. Miller, M.D., Merck: Grant/Research Support|UptoDate: Royalties José M Munita, MD, MSD: Grant/Research Support|Pfizer: Grant/Research Support

Infections due to antimicrobial-resistant Gram-negative organisms present increasingly difficult therapeutic challenges, especially in the presence of metallo-β-lactamases. We present the case of a patient with cholangitis due to Pseudomonas aeruginosa and Klebsiella pneumoniae isolates that developed cefiderocol resistance on therapy treated successfully with cefepime-zidebactam. Serial clinical isolates recovered from biliary fluid and ascitic fluid were tested for susceptibility to cefiderocol, aztreonam-avibactam, cefepime-taniborbactam, cefepime-zidebactam, and cefiderocol-xeruborbactam by broth microdilution. Whole-genome sequencing was performed to identify resistance determinants. An emergency investigational new drug application was authorized by the United States Food and Drug Administration for the compassionate use of cefepime-zidebactam based on susceptibility test results. Index isolates of P. aeruginosa (IMP positive) and K. pneumoniae (NDM-5, OXA-232 positive) tested susceptible to cefiderocol by disk diffusion in the clinical microbiology laboratory. The patient was treated with a regimen of cefiderocol and eravacycline, with persistent fever and development of hepatic microabscesses on imaging. Compassionate use cefepime-zidebactam therapy was initiated the day prior to liver transplantation and continued for a total of 14 days due to positive ascitic fluid cultures obtained during the operation. The K. pneumoniae and P. aeruginosa were cefiderocol resistant by broth microdilution. Cefepime-zidebactam remained active with MICs of 8/8 mg/L and 32/32 mg/L, respectively. The patient did well post-transplant and resumed chemotherapy. Antimicrobial therapy with cefepime-zidebactam along with source control allowed successful liver transplantation in a patient with cefiderocol-resistant K. pneumoniae and P. aeruginosa. Cefepime-zidebactam may be a therapeutic option for extensively drug-resistant Gram-negative organisms.

Background:Infections due to antimicrobial-resistant Gram-negative organisms present increasingly difficult therapeutic challenges, especially in the presence of metallo-β-lactamases. We present the case of a patient with cholangitis due to Pseudomonas aeruginosa and Klebsiella pneumoniae isolates that developed cefiderocol resistance on therapy treated successfully with cefepime-zidebactam. Methods:Serial clinical isolates recovered from biliary fluid and ascitic fluid were tested for susceptibility to cefiderocol, aztreonam-avibactam, cefepime-taniborbactam, cefepime-zidebactam, and cefiderocol-xeruborbactam by broth microdilution. Whole-genome sequencing was performed to identify resistance determinants. An emergency investigational new drug application was authorized by the United States Food and Drug Administration for the compassionate use of cefepime-zidebactam based on susceptibility test results. Results:Index isolates of P. aeruginosa (IMP positive) and K. pneumoniae (NDM-5, OXA-232 positive) tested susceptible to cefiderocol by disk diffusion in the clinical microbiology laboratory. The patient was treated with a regimen of cefiderocol and eravacycline, with persistent fever and development of hepatic microabscesses on imaging. Compassionate use cefepime-zidebactam therapy was initiated the day prior to liver transplantation and continued for a total of 14 days due to positive ascitic fluid cultures obtained during the operation. The K. pneumoniae and P. aeruginosa were cefiderocol resistant by broth microdilution. Cefepime-zidebactam remained active with MICs of 8/8 mg/L and 32/32 mg/L, respectively. The patient did well post-transplant and resumed chemotherapy. Conclusion:Antimicrobial therapy with cefepime-zidebactam along with source control allowed successful liver transplantation in a patient with cefiderocol-resistant K. pneumoniae and P. aeruginosa. Cefepime-zidebactam may be a therapeutic option for extensively drug-resistant Gram-negative organisms.

Once considered rare in eukaryotes, polycistronic mRNA expression has been identified in kinetoplastids and, more recently, green algae, red algae, and certain fungi. This study provides comprehensive evidence supporting the existence of polycistronic mRNA expression in the apicomplexan parasite Cryptosporidium parvum. Leveraging long-read RNA-seq data from different parasite strains and using multiple long-read technologies, we demonstrate the existence of defined polycistronic transcripts containing 2-4 protein encoding genes, several validated with RT-PCR. Some polycistrons exhibit differential expression profiles, usually involving the generation of internal monocistronic transcripts at different times during development. ATAC-seq in sporozoites reveals that polycistronic transcripts usually have a single open chromatin peak at their 5-prime ends, which contains a single E2F binding site motif. Polycistronic genes do not appear enriched for either male or female exclusive genes. This study elucidates a potentially complex layer of gene regulation with distinct chromatin accessibility akin to monocistronic transcripts. This is the first report of polycistronic transcription in an apicomplexan and expands our understanding of gene expression strategies in this medically important organism.

Abstract Background Cefiderocol (FDC) is a last-line agent used to treat carbapenem-resistant P. aeruginosa (CR-PA). We evaluated the prevalence of FDC heteroresistance (hR) and non-susceptibility (NS) in the POP cohort and associations with genes previously related to FDC resistance.Figure 1.Proportion of isolates harboring genomic feature by geographic region (US vs non-US). Methods 972 genomes from the multicenter, global POP study were screened for mutations in genes implicated in decreased FDC susceptibility: ampC, pirR, pirS, pirA, piuA/D, ftsI, cpxS and exogenous β-lactamases. A representative population was selected to encompass all variants as well as controls obtained by phylogenetically mapping each mutant isolate with a closest non-mutant isolate (n=187). All strains were assessed by broth microdilution (BMD) and population analysis profile (PAP). FDC NS was defined as minimum inhibitory concentration (MIC) ≥ 8 μg/m. hR was determined by PAP area under the curve > 80, per the 99% confidence interval for PAO1. Chi-squared test was used to assess correlation of ceftolozane-tazobactam (C/T), ceftazidime-avibactam (CZA), and imipenem-relebactam (IMR) categorization and FDC hR/NS as well as gene correlations with FDC hR/NS and geographic region of the isolate. Association between FDC phenotype and MIC category of novel β-lactam/β-lactamase inhibitor combinations. Results Over 20% of isolates displayed a NS or hR FDC phenotype (n=39: 28 hR, 11 MIC ≥ 8 μg/mL), equally distributed among US and non-US isolates (Fig 1, P =0.36). Isolates with increased C/T, CZA, and IMR MICs were more likely to be FDC NS or hR (Tbl 1). IMR susceptibility was low across FDC NS, hR and susceptible cohorts. The combination of mutations in both ampC and the PirRS system demonstrated higher risk for FDC NS or hR (Tbl 2, P = 0.0003), but isolated mutations in either ampC or PirRS did not increase risk for FDC NS or hR. VEB and NDM β-lactamases were present in non-US isolates and their presence correlated with FDC NS or hR (Tbl 2, P = 0.0003, 0.0017). Mutations in the PirRS system showed a geographic predisposition in US isolates (Fig 1, P= 0.0047), while exogenous β-lactamases were associated with international isolates (P < 0.0001). Genotypic determinants associated with FDC susceptibility phenotype. Conclusion NS and hR to FDC is prevalent globally among CR-PA and is associated with reduced C/T, CZA, and IMR susceptibility. NS and hR isolates were associated with mutations in ampC, pirRS, and the exogenous β-lactamases VEB, NDM. Further studies are needed to define clinical significance of FDC hR. Disclosures Cesar A. Arias, MD, MSc, PhD, UpToDate, Inc.: Royalties Vincent Tam, Pharm. D., AbbVie Inc: Advisor/Consultant Michael J. Satlin, MD, AbbVie: DSMB participant|bioMerieux: Grant/Research Support|Merck: Grant/Research Support|Selux Diagnostics: Grant/Research Support|SNIPRBiome: Grant/Research Support William R. Miller, M.D., Merck: Grant/Research Support|UptoDate: Royalties

Abstract Background In the intensive care unit (ICU), Candida spp. gut colonization is a risk factor for Candida bloodstream infection; however, the specific risk factors for Candida gut colonization remain unknown. Impaired anti-microbial immune responses may predispose to Candida gut colonization. LL37, an antimicrobial peptide (AMP) from the cathelicidin family, plays an important role in the immune response to pathogens, including Candida. The purpose of this study was to determine whether plasma LL37 levels are associated with Candida gut colonization in the ICU. Figure 1. Plasma LL-37 levels comparing patients with (Candida=1) and without (Candida=0) Candida gut colonization. Methods We examined a sample of patients enrolled in DYNAMITE (a prospective cohort study of ICU patients at a tertiary care hospital) who provided stool samples. Clinical data was collected by chart review. The first stool sample collected after enrollment was diluted in saline and plated on selective media for Candida spp.; species was identified from positive samples via matrix-assisted laser desorption/time-of-flight. Plasma samples were analyzed for LL37 levels via enzyme-linked immunosorbent assay. LL37 levels were normalized with a four-parameter logistic curve. We compared plasma LL37 levels between patients with and without Candida gut colonization, as well as different colonizing Candida species, using an unpaired t-test in R. Figure 2. Plasma LL-37 levels comparing patients with different Candida species causing gut colonization (0=no Candida gut colonization; 1=C. albicans; 2=C. glabrata; 3=C. parapsilosis; 4=C. albicans/C. glabrata co-colonization). Results Of 31 patients included, 14 (48%) had Candida gut colonization (6 [19%] C. albicans; 5 [16%] C. glabrata; 2 [6%] C. glabrata and C. albicans; 1 [3%] C. parapsilosis). Mean age was 60±16 years; 22 (71%) patients were white; 16 (52%) were women; and 10 (32%) were in shock on ICU admission. There was no statistically significant difference in plasma LL37 between patients with and without Candida gut colonization (mean 44.9± 22.6 ng/mL vs. mean 42.0 ±18.8 ng/mL, respectively, p=0.71) (Figure 1). LL37 levels also did not differ according to colonizing Candida species (Figure 2). Conclusion In this pilot study, plasma LL37 was not found to be associated with Candida gut colonization among ICU patients. These findings suggest that systemic levels may not reflect tissue (intestinal) immune responses. Understanding the role of LL37 and other AMPs in Candida colonization may inspire alternative approaches to reduce Candida colonization and resultant Candida bloodstream infections in the ICU. Disclosures All Authors: No reported disclosures

Half the world's population is at risk of developing a malaria infection, which is caused by parasites of the genus Plasmodium. Currently, resistance has been identified to all clinically available antimalarials, highlighting an urgent need to develop novel compounds and better understand common mechanisms of resistance. We previously identified a novel tetrahydro-β-carboline compound, PRC1590, which potently kills the malaria parasite. To better understand its mechanism of action, we selected for and characterized resistance to PRC1590 in Plasmodium falciparum. Through in vitro selection of resistance to PRC1590, we have identified that a single-nucleotide polymorphism on the parasite's multidrug resistance protein 1 (PfMDR1 G293V) mediates resistance to PRC1590. This mutation results in stereospecific resistance and sensitizes parasites to other antimalarials, such as mefloquine, quinine, and MMV019017. Intraerythrocytic asexual stage specificity assays have revealed that PRC1590 is most potent during the trophozoite stage when the parasite forms a single digestive vacuole (DV) and actively digests hemoglobin. Moreover, fluorescence microscopy revealed that PRC1590 disrupts the function of the DV, indicating a potential molecular target associated with this organelle. Our findings mark a significant step in understanding the mechanism of resistance and the mode of action of this emerging class of antimalarials. In addition, our results suggest a potential link between resistance mediated by PfMDR1 and PRC1590's molecular target. This research underscores the pressing need for future research aimed at investigating the intricate relationship between a compound's chemical scaffold, molecular target, and resistance mutations associated with PfMDR1.

Multiple displacement amplification (MDA) outperforms conventional PCR in long fragment and whole-genome amplification, making it attractive to couple MDA with long-read sequencing of samples with limited quantities of DNA to obtain improved genome assemblies. Here, we explore the efficacy and limits of MDA for efficient low-cost genome sequence assembly using Oxford Nanopore Technologies (ONTs) rapid library preparations and minION sequencing. We successfully generated almost complete genome sequences for all organisms examined, including Gram-positive (Staphylococcus aureus, Enterococcus faecium) and Gram-negative (Escherichia coli) prokaryotes and one challenging eukaryotic pathogen (Cryptosporidium spp) representing a broad spectrum of critical infectious disease pathogens. High-quality data from those samples were generated starting with only 0.025 ng of total DNA. Controlled sheared DNA samples exhibited a distinct pattern of size increase after MDA, which may be associated with the amplification of long, low-abundance fragments present in the assay, as well as generating concatemeric sequences during amplification. To address concatemers, we developed a computational pipeline (CADECT: Concatemer Detection Tool) to identify and remove putative concatemeric sequences. This study highlights the efficacy of MDA in generating high-quality genome assemblies from limited amounts of input DNA. Also, the CADECT pipeline effectively mitigated the impact of concatemeric sequences, enabling the assembly of contiguous sequences even in cases where the input genomic DNA was degraded. These results have significant implications for the study of organisms that are challenging to culture in vitro, such as Cryptosporidium, and for expediting critical results in clinical settings with limited quantities of available genomic DNA.

Abstract Background Critically ill patients have frequent colonization with multi-drug resistant organisms (MDRO). Colonization is assumed to be a static state, but intensive care unit (ICU) patients may be exposed to various interventions that affect their colonization status; this has not been rigorously examined.Table 1.Baseline characteristics of intensive care unit patients by multidrug-resistant stool colonization status. Methods We performed a prospective cohort study of adult ICU patients. Patients had stool samples collected twice weekly for ≤ 4 weeks or until ICU discharge; samples were plated on selective media for the MDROs vancomycin-resistant enterococci (VRE), extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E), and carbapenem-resistant Enterobacterales (CRE). We characterized patient demographics and changes in colonization status and compared outcomes between patients with and without MDRO colonization using a desirability of outcomes ranking (DOOR) analysis, which considered three outcome levels (alive, alive with infection, or dead).Figure 1.Changes in multi-drug resistant stool colonization status over time for 200 patients admitted to an intensive care unit. Results We included 200 patients; 82 (41%) were colonized at ≥1 time point; 50 (25%) had ≥ 50% of their samples colonized; 32 (16%) had ≥ 2 consecutive samples with colonization (“persistent”). There were no differences in baseline characteristics between patients with and without any colonization (Table 1), or between patients with or without ≥ 50% or persistent colonization. We observed flux in colonization status (Figure 1); for example, of 53 patients colonized on day 1, 25 (47%) were not colonized on day 7. Of 119 patients with multiple samples, 38 (32%) had changes in their colonization status from first to last sample (Figure 2). Any colonization or ESBL/CRE colonization was not associated with adverse outcomes. However, patients with VRE colonization had worse DOOR outcomes than patients without (58.4% probability of a worse DOOR outcome, 95% CI 50.3%-66.2%) (Figure 3).Figure 2.Changes in multi-drug resistant stool colonization status between first and last sample for 119 patients who had multiple samples. Conclusion Over 40% of ICU patients were colonized with VRE, ESBL-E, and/or CRE, although colonization status commonly changed over time. Colonization status detected at one timepoint (e.g., ICU admission) may not reflect future states. More research is needed to identify factors that facilitate persistence and loss of colonization, and how this influences outcomes.Figure 3.Desirability of outcome ranking (DOOR) levels comparing intensive care unit patients without (top bar) and with (bottom bar) VRE stool colonization. Disclosures All Authors: No reported disclosures

Abstract Background Cefazolin is an alternative for the treatment of MSSA infections and first choice option for surgical prophylaxis. The cefazolin inoculum effect (CzIE), the increase in the MIC at high bacterial inocula is associated to therapeutic failures and increased mortality in deep-seated MSSA infections. The prevalence of the CzIE among nasal colonizing MSSA from ICU patients is currently unknown. The aim of this study was to investigate the CzIE in nasal MSSA recovered from patients admitted to ICUs in Colombia. Methods We evaluated 33 MSSA isolates recovered from nasal swabs of 29 patients admitted to ICU at 6 high-complexity Colombian hospitals (2019-2023). CFZ MIC using gold standard broth microdilution at high inoculum was done. The modified nitrocefin-based rapid test to identify the CzIE was performed, and diagnostic performance metrics were calculated. Whole genome sequencing was used to characterize BlaZ types and allotypes, Clonal Complexes CC and Accessory Gene Regulator (Agr) types. Results The CzIE was identified in 54.5% of nasal MSSA isolates. A high prevalence of nasal colonization with MSSA exhibiting the CzIE was detected (59%, 17 patients). Among 18 MSSA with the CzIE, type A BlaZ was the predominant β-lactamase (61%) and BlaZ-2 was the most common allotype (50%). Whereas among 15 MSSA lacking the CzIE, BlaZ type C was the most frequent (47%) and BlaZ-1 was the allotype predominant (40%). A high diversity of genetic lineages with eight CC was detected among the 33 MSSA. CC30 was the predominant lineage in MSSA displaying the CzIE (44%), while CC5 and CC30 (27% each one) were the most common in MSSA without the CzIE. Agr-III and Agr-I were predominant Agr types in 56% and 47% of MSSA with the CzIE and lacking the CzIE, respectively. Compared to the gold standard the modified rapid test identified nasal MSSA isolates showing the CzIE with a sensitivity of 100%, specificity of 93% and an overall accuracy of 97%. Conclusion We found an unexpected high prevalence (59%) of ICU patients colonized by MSSA exhibiting the CzIE, showing similar genetic features to MSSA with CzIE from invasive infections. Furthermore, our findings suggest that the nasal cavity of ICU patients may represent a reservoir for the dissemination of MSSA showing the CzIE, highlighting the importance of screening and surveillance. Disclosures All Authors: No reported disclosures

Background Candida auris (C auris) has rapidly spread in the United States. We aimed to characterize the trends in volumes and sources of clinical cultures with C auris at a large health care system. Methods We conducted a retrospective observational study including clinical cultures with C auris collected between April 1, 2019, and December 31, 2023. Surveillance cultures were excluded. Clinical specimens were processed through routine methods, and identification was performed using mass spectrometry. Whole-genome sequencing was performed on select specimens. Results We identified 327 clinical cultures belonging to 231 unique patients. The number of clinical cultures increased each year, from 5 in 2019 to 29 in 2020 (580%), 71 in 2021 (251% relative to 2020), 107 in 2022 (46% relative to 2021), and 115 in 2023 (7% relative to 2022). Blood cultures were the most common source, but specimens originating from soft tissue/bone infections had a large increase in 2022 and 2023. All sequenced isolates belong to clade III (South African clade) and were resistant to fluconazole and susceptible to echinocandins and amphotericin B. Conclusions The volumes of clinical cultures with C auris have rapidly increased, accompanied by an expansion in the sources of infection.

Abstract Background Acinetobacter baumannii (AB) is known to have high rates of multidrug resistance (MDR), new treatment options are critically needed to prevent further spread and mortality. Deferiprone (DFP), a metal chelator, has been shown to induce transient growth inhibition in previous studies, and this could be potentially exploited for therapeutic purposes. The objective of the study was to examine the impact of iron limitation on the in vitro and in vivo activity of ceftazidime against AB. Methods An MDR bloodstream isolate (AB14) recovered from a 50 years-old male patient was used. Ceftazidime (CAZ) minimal inhibitory concentration was determined by microbroth dilution in the presence of DFP. In vitro growth profiles were discerned by an automated process (BacterioScan 216Dx) tracking CFU/mL over time. Bacteria were started at a concentration of approximately 5.5 log CFU/mL in tryptic soy broth. The effect of DFP (1000μM) and CAZ (128μg/ml) on bacterial growth was studied either alone or in combination. A neutropenic pneumonia mouse model was used in which the animals were given two doses of cyclophosphamide and one dose of uranyl nitrate intraperitoneally prior to infection. For infection, anesthetized mice were inoculated with approximately 1 × 106 CFU (with and without DFP) via the trachea under laryngoscopic guidance. A humanized regimen consisting of three doses of CAZ over 8h was used to mimic a dose of 2g. Quantitative culture was used to ascertain the bacterial burden of lung tissue immediately and 8h post infection. Results MIC of CAZ was reduced from > 256μg/mL to 64, 32, and 1μg/mL in the presence of 500, 1000, and 1500μM of DFP respectively. A combination of subinhibitory concentrations of CAZ and DFP resulted in minimal (< 0.5 log CFU/mL) growth in 20h. The lung bacterial burden of the control groups increased ≥ 1 log CFU/g after 8h, whereas the combination group (CAZ + DFP) stayed comparable to baseline. Control groups compared to combination was statistically different p< 0.05). Conclusion These results support that limiting iron has the potential to be a novel approach to treating MDR AB infections. Further research is warranted to broaden the scope to encompass additional clinical isolates, and to optimize the approach as a therapeutic solution. Disclosures All Authors: No reported disclosures