bluuebunny/crossref_metadata_2025_split huggingface dataset:
2160. Emergence of INDEL Mutations in KPC-2: Insights into Ceftazidime-Avibactam and Imipenem-Relebactam Efficacy
Authors: Brunetti Florencia , Bethel Christopher R , Sanz María Belén , Pasterán Fernando , Gutkind Gabriel , Gomez Sonia , Bonomo Robert A , Power Pablo 
Published: November 2023

Abstract

Background
Enterobacterales producing KPC-2 and KPC-3 β-lactamases (Bla) are a significant clinical threat. Ceftazidime/avibactam (CZA) and imipenem/relebactam (IMI/REL) are successful in the treatment for these infections. Unfortunately, CZA-resistant isolates are emerging as a result of amino acid changes in three “hotspots” in the KPC Bla: Ω-loop region; the 237-243 loop; and the 266-275 loop (Figure 1). Our goal here is to assess the effect of insertion-deletion (INDEL) mutations located in the three hotspots in KPC-2, and their impact on the efficacy of these two drug combinations

Methods
The blaKPC-44 (ins_261_AVYTRAPNKDDKHSE), blaKPC-80 (ins_267_PNK), blaKPC-81 (del_173_I), blaKPC-97 (ins_276_VNSEA), and blaKPC-160 (del_167_LE) genes were amplified by PCR from K. pneumoniae isolates resistant to CZA. The blaKPC-14 (del_242-243_GT) gene was obtained by mutagenesis from the blaKPC-2 gene. Genes were cloned in pMBLe and transformed in E. coli TOP10 for MIC determinations. Immunoblotting with polyclonal anti-KPC-2 antibody was performed in whole-cell preparations. Molecular modeling of KPC variants were obtained by Yasara

Results
All KPC variants demonstrated lower MICs compared to KPC-2 for most β-lactams (carbapenems included), except for CAZ, where variants exhibited up to 8-fold higher MICs. All variant clones showed at least a 3-dilution higher MICs for CZA, except KPC-160. IMI-REL restored susceptibility to KPC-2 and INDEL variants. The Ω−loop variants exhibited lower MICs compared to the other hotspots variants (CZA included). INDEL mutations resulted in decreased levels of protein production, with KPC-81 production being the most affected. In silico models suggest that INDEL mutations have different effects depending on the structural domain affectedTable 1.Minimum Inhibitory Concentrations (μg/mL) of E. coli TOP10F´ recombinant clones

Conclusion
INDEL mutations in the three hotspots of KPC-2 can impair CZA activity, but not affect IMI/REL susceptibility. The differences in MICs among the variants suggest that each hotspot affects the interactions of KPC-2 with CAZ and AVI through different mechanisms. Additionally, INDEL mutations appear to decrease protein stability as Bla production levels are reduced. This work provides relevant information about the changes in the interaction with DBO inhibitors associated with INDEL mutations in KPC-2

Disclosures
Robert A. bonomo, MD, Entasis, Merck, VenatoRx, Wockhardt: Grant/Research Support
2940. Weaving Federal and Private Funding to Achieve Hepatitis C Elimination Goals at Syringe Services Programs in Rural Kentucky
Authors: Van Sickels Nicholas , Soria Jaime , Thornton Alice C , Wilburn Amanda B , Collins Jana 
Published: November 2023

Abstract

Background
Addressing the syndemic of HIV, Hepatitis C (HCV), and Opioid Use Disorder in rural Kentucky requires unique approaches to funding, assessment, and treatment. The Kentucky Income Reinvestment Project (KIRP) leverages Ryan White, CDC, and private funding to provide harm reduction services, including HIV and Hepatitis C testing, across the majority of Kentucky's Syringe Services Programs (SSPs). We sought to compare HCV evaluations between SSPs with KIRP support versus SSPs without KIRP support. Additionally, we describe early results from a pilot study providing direct treatment at the SSP from 2 KIRP-supported counties (Figure 1).

Methods
We performed a cross-sectional review of data reported to Kentucky Department for Public Health from March 2021 to March 2023. OraQuick® HCV rapid antibody tests were used for screening. We compared the number of eligible clients seen and tested for HCV, the seropositivity, and referral rates for treatment. Additionally, we report pilot outcomes from two counties, where patients were treated at the SSP. Study data was managed using the REDCap data capture tool. Chi-squared tests were used to describe statistical differences between groups, with analyses performed using Stata 17 (College Station, TX: Stata Corp LLC).

Results
Over a 24-month period, KIRP sites saw and tested a higher volume and percentage of clients than non-KIRP sites (18.3% versus 4.9%, p&lt; 0.001; see Figure 2). HCV seropositivity was similar between KIRP and non-KIRP sites (31.5% versus 23.2%, p=0.058). HCV treatment referrals were greater at KIRP sites, with 646/654 (98.8%) referred compared to 26/29 (89.7%) referred at non-KIRP sites (p=0.009). Pilot counties received 107 referrals from May 2022 to March 2023. 51/107 were evaluated; nine had negative RNA testing, 17 did not show, and 25 completed one visit. To date, 5 patients achieved SVR, and 5 are undergoing treatment (Figure 3).

Conclusion
Our study demonstrates significantly higher rates of HCV testing, linkage, and referral at KIRP-supported SSPs in Kentucky. Overlaying funding from the Ryan White Care Act with CDC and private entities allows for status neutral assessments for HIV and HCV. Partnerships for direct treatment of HCV at the SSP, especially in more rural areas, are crucial to the HCV elimination strategy.

Disclosures
All Authors: No reported disclosures
2169. A 4-Year Analysis of Antimicrobial Susceptibility to Ceftolozane/Tazobactam in Pseudomonas aeruginosa: How Drug Availability Affects Resistance Rate
Authors: Imeneo Alessandra , Campogiani Laura , Di Lorenzo Andrea , D’Agostini Cartesio , Altieri Anna , Malagnino Vincenzo , Andreoni Massimo , Iannetta Marco , Sarmati Loredana 
Published: November 2023

Abstract

Background
Antibiotic resistance is associated with a fitness cost and reduced bacterial growth that allows susceptible bacteria to outcompete resistant bacteria, if selective pressure from antibiotics is reduced. Ceftolozane/tazobactam (C/T) approved in 2014, was temporarily withdrawn from December 2020 to February 2022: this forced unavailability created the conditions to study how drug discontinuation might influence resistance reversibility.

Methods
This is a retrospective observational study, including isolates of P. aeruginosa collected between March 1st 2019 and February 22nd 2023. Isolates collected from swabs (e.g. rectal, skin) or with no C/T susceptibility test were excluded. Antibiotic susceptibility tests were performed with the Vitek-2 assay. Only the first isolate for each patient was included; multiple isolates from the same patient were included if collected at least 5 months apart. Four collection periods were defined (Fig 1). All data were analyzed using JASP.

Results
From the Hospital microbiology laboratory list, 1768 P. aeruginosa isolates were derived, of which 926 isolates fulfilled the inclusion criteria. A significant (p-value &lt; 0.001) reduction of C/T resistance rate was observed when the antibiotic was unavailable, followed by a subsequent increase with its reintroduction (Tab.1). Evaluating phenotypic susceptibility to other antibiotics, production of GES-variants, suggested by resistance to carbapenem and C/T but retained susceptibility to ceftazidime/avibactam, was most likely the predominant resistance mechanism (Tab. 2); a reduction in GES-like isolates, concurrent with decreasing C/T resistance was observed. 19 patients presented a C/T-resistant isolate after a previous susceptible one, but only 4 patients had received a prior C/T treatment (Tab 3).

Conclusion
The temporary unavailability of C/T created the condition to analyze the practical application of the theory of fitness cost to maintain resistance. Our data show a significant reduction of resistance in the absence of the antibiotic, but a subsequent increase with its reintroduction, due to the persistence of resistant isolates. Therefore, continuous monitoring of antibiotic use and evolving resistance is essential.

Disclosures
All Authors: No reported disclosures
2171. Minocycline Susceptibility in Carbapenem-Resistant Acinetobacter baumannii Blood Isolates in South Korea: Role of tetB Gene in Resistance
Authors: Kim Taeeun , Jun Eun Hee , Hong Yoon-Kyoung , Jung Jiwon , Kim Min Jae , Sung Heungsup , Kim Mi-Na , Kim Sung-Han , Choi Sang-Ho , Lee Sang-Oh , Kim Yang Soo , Chong Yong Pil 
Published: November 2023

Abstract

Background
Carbapenem-resistant Acinetobacter baumannii (CRAB) represents a growing global threat with minimal therapeutic options. Minocycline (MIN), a semisynthetic tetracycline derivative, has been suggested as an attractive therapeutic option for CRAB infection. However, the optimal dosing and breakpoint for MIN in treating CRAB infection remain unclear. Furthermore, nonsusceptibility to MIN may occur through the efflux pump, in particular, TetB. As the prevalence of tetB in A. baumannii has increased, so have the MIN minimum inhibitory concentrations (MICs) for A. baumannii strains carrying tetB. Here, we evaluated the MIN susceptibility rate in CRAB clinical strains and the association between tetB carriage and MIN susceptibility.

Methods
Representative CRAB blood isolates were collected from a tertiary care center in South Korea. The Clinical and Laboratory Standards Institute (CLSI) MIN susceptibility breakpoint for A. baumannii was defined as MIC ≤4 mg/L, whereas a new MIN breakpoint of 1 mg/L was suggested based on pharmacokinetic (PK)/pharmacodynamic (PD) studies. For comparison of the impact of the tetB resistance, tigecycline was used. Tigecycline susceptibility breakpoint for Enterobacterales defined by EUCAST was applied (≤0.5 mg/L). TetB carriage was detected by polymerase chain reaction.

Results
Of 160 CRAB isolates, 134 (84%) were susceptible to MIN according to the CLSI criteria, among which 40% (53/134) were PK-PD non-susceptible. The MIN MIC 50/90 was 1/8 mg/L. Seventy-nine (49%) isolates carried tetB. One-third (26/79) of tetB-positive and none of the tetB-negative isolates were CLSI non-susceptible, while 66% of tetB-positive and 33% of tetB-negative isolates were PK-PD non-susceptible. TetB carriage was correlated with a higher MIN MIC than non-tetB carriage (MIC 50/90 2/8 mg/L vs. 1/2 mg/L) (Figure 1). However, no clear correlation was observed between tetB-positivity and tigecycline MIC (Figure 2). Overall, tetB positivity demonstrated 100% sensitivity for MIN CLSI non-susceptibility and 66% sensitivity for PK-PD non-susceptibility.

tetB positivity and MICs of minocycline for carbapenem-resistant A. baumannii. The CLSI susceptibility breakpoint for minocycline is ≤4 mg/L, whereas a new MIN PK-PD breakpoint of 1 mg/L was suggested.
Figure2.
tetB positivity and MICs of tigecycline for carbapenem-resistant A. baumannii. The tigecycline susceptibility breakpoint for Enterobacterales defined by EUCAST was applied (≤0.5 mg/L).

Conclusion
In our study, 49% CRAB isolates carried tetB gene. Our findings suggest that the lack of tetB is not a reliable marker of MIN PK-PD susceptibility. Therefore, further clinical data on MIN usage is necessary.

Disclosures
All Authors: No reported disclosures
2175. Emergence of Dual-Carbapenemase Producing Enterobacterales in Chile After the COVID-19 Pandemic
Authors: Villamil Aura , Rodas Paula I , Quesille-Villalobos Ana M , Martinez Jose R W , Alcalde-Rico Manuel , Quiroz Valeria E , Rivas Lina M , Riquelme María P , Solar Camila , Rafael Araos , Dias Lorena , Garcia Patricia , Munita José M 
Published: November 2023

Abstract

Background
Carbapenem-resistant Enterobacterales (CRE) are a major public health threat, largely due to the presence of carbapenemases, which are globally disseminated in mobile genetic elements. The emergence of CRE carrying multiple carbapenemases has been reported in several countries, particularly after the COVID-19 pandemic. In this study, we report the emergence of dual-producing CRE (DP-CRE) in Chile and provide a phenotipic and genomic characterization using whole-genome sequencing (WGS).

Methods
We evaluated the presence of carbapenemase in a total of 1367 CRE isolates recovered from invasive infections in 11 healthcare centers since 2018. Among them, 9 DP-CRE were detected and included in this report. Antimicrobial susceptibility was tested by broth microdilution and disk diffusion methods (CLSI, 2023), while blaKPC, blaVIM, blaIMP and blaNDM genes were detected by PCR. WGS was carried out using short and long reads (Illumina and Oxford Nanopore) and hybrid assemblies were performed.

Results
All 9 DP-CRE identified were recovered between November 2021 and June 2022 from 3 healthcare centers of a single city. In terms of species, 6 were identified as E. coli, one isolate of K. pneumoniae, one K. oxytoca and one C. freundii. All DP-CRE identified carried the combination of blaKPC and blaNDM. Genomic analyses confirmed all but one isolate carried blaKPC-2 and blaNDM-7. The remaining genome belonged to a K. pneumoniae that harboured blaKPC-3 and blaNDM-7. All 9 isolates exhibited resistance to all β-lactams, including carbapenems, aztreonam (ATM), cephalosporins and β-lactam/β-lactamase inhibators. Cefiderocol (FDC) was the only compound active against all the isolates. Also, all the DP-CRE became susceptible to ATM when combined with ceftazidime/avibactam (CZA). Hybrid assemblies revealed that blaKPC and blaNDM were harboured on independent plasmids (∼58,900 bp and ∼41,100 bp, respectively) as shown in Figure 1.

Conclusion
To the best of our knowledge, this is the first report of the emergence of DP-CRE in Chile after COVID-19 pandemic. Our results highlight the relevance of active surveillance of multidrug-resistance pathogens. FDC and CZA/ATM were the only compounds that remained active in vitro against these pathogens.

Disclosures
All Authors: No reported disclosures
2176. Cardiolipin Synthases Play Redundant Roles And Are Required for Membrane Remodeling in Daptomycin Resistant Enterococcus faecalis
Authors: Nguyen April , Tran Truc , Panesso Diana , Hood Kara S , Polamraju Vinathi , Zhang Rutan , Khan Ayesha , Miller William R , Mileykovskaya Eugenia , Shamoo Yousif , Xu Libin , Vitrac Heidi , Arias Cesar A 
Published: November 2023

Abstract

Background
Daptomycin (DAP) is a lipopeptide antibiotic targeting anionic phospholipids (APLs) at the division septum. Resistance to DAP (DAP-R) in E. faecalis (Efs) has been associated with activation of the LiaFSR response and redistribution of APL microdomains through the effector protein LiaX. We also identified a novel transmembrane protein (designated LiaY) as essential for changes in APL microdomains which contain cardiolipin (CL). Efs encodes two CL synthase genes, cls1 and cls2. While changes in Cls1 are associated with DAP-R, their link to the LiaFSR system remains unclear. We aim to establish the roles of Cls in DAP-R to provide insights into membrane remodeling associated with DAP-R.

Methods
qRT-PCR was used to study cls expression. Membrane lipid content was analyzed using hydrophilic interaction chromatography-mass spectrometry. Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test and localization of APL microdomains with 10-N-nonyl-acridine orange. The bacterial two hybrid system was used to evaluate protein interaction. Fluorescence microscopy evaluated protein co-localization.

Results
cls1 was upregulated in DAP-R Efs OG117ΔliaX. Deletion of either cls caused upregulation of the remaining cls gene. Development of DAP-R altered membrane lipid content, namely, an increase in CL and a shift towards species with longer fatty acid chains and higher degree of unsaturation. Upon deletion of both cls, CL content was eliminated, peripheral APL microdomains were abolished, and a decrease in the DAP MIC was observed. LiaY interacted with Cls1, but not Cls2 using the two-hybrid system. The interaction was confirmed with fluorescence microscopy showing co-localization of LiaY and Cls1 at the septum and away from the septum in DAP-S and DAP-R strains, respectively. LiaY and Cls1 were also individually co-localized to APL microdomains.

Conclusion
While Cls1 is predominantly associated with DAP-R, we show a functional redundancy between Cls1 and Cls2 in both membrane homeostasis and in DAP-R. Cls1 interacts with LiaY, which bridges the resistance mechanism with membrane adaptation, ultimately leading to altered localization of APL microdomains that divert DAP away from the division septum.

Disclosures
William R. Miller, M.D., Merck: Grant/Research Support|UpToDate: Honoraria
2178. Resistance to Cefepime/Taniborbactam in NDM-1 -Are We Losing the Battle against MDROs?
Authors: Ono Daisuke , Mojica Maria F , Bethel Christopher R , Ishii Yoshikazu , Moreno Diego , Vila Alejandro J , Bonomo Robert A 
Published: November 2023

Abstract

Background
Taniborbactam (TAN; Fig 1) is a novel cyclic boronate that inhibits Serine- and some Metallo-β-Lactamases like NDM and VIM. The combination cefepime (FEP)/ TAN has just completed a Phase III clinical trial. Notably, resistance mechanisms of FEP/TAN have already been described, such as the one provided by NDM-9 (Glu152Lys; PMID:36796395). The crystal structure of the NDM-1:TAN complex revealed that Lys224 anchors TAN by forming two hydrogen bonds (PMID:31454231). We previously showed that the Arg228Leu substitution in VIM-24, the homologous residue of Lys224 in VIM-2, improves the activity towards ceftazidime (CAZ) and FEP (PMID:25915520 ). Therefore, we hypothesized that substitutions at position 224 in NDM-1 could also have a similar effect and potentially confer FEP/TAN resistance.

Methods
NDM-1 clones containing all 19 amino acid variants at position 224 were synthesized (Genscript, NJ) and used to transform E. coli DH10B. Minimal inhibitory concentrations (MICs) were determined using the CLSI-approved agar dilution method. NDM-1 and Lys224Ile were further studied by steady-state kinetics, inhibitor analysis, and dockings.

Results
Compared to NDM-1, all but the Lys224Ile variant display lower MICs for FEP, CAZ, and imipenem (IMI) and higher MICs for FEP/TAN. Contrary, resistance to FEP and CAZ conferred by the Lys224Ile variant was similar to that by NDM-1. However, TAN did not alter MIC for FEP (Table 1). Kinetic assays confirmed differences in the catalytic efficiency of NDM-1 and the Lys224Ile variant to FEP and IMI, respectively (Table 2). Likewise, the IC50 of TAN for NDM-1 (0.01 µM) was significantly lower than for the Lys224Ile variant (0.14 µM) (Table 2). Lastly, docking simulations revealed that the Lys224Ile substitution results in the loss of the anchoring H-bonds and yields an unproductive binding of TAN (Fig 2-a, b).

Conclusion
Resistance to CEF/TAN arises from the Lys224Ile substitution in NDM-1. This finding, in addition to the occurrence of NDM-9 (Glu152Lys), may portend vulnerabilities in this novel β-lactam/β-lactamase inhibitor combination by single amino acid changes even before clinical release. Further attention should be focused on exploring the impact of other amino acid substitutions on susceptibility to CEF/TAN to anticipate clinical failure.

Disclosures
Robert A. bonomo, MD, Entasis, Merck, VenatoRx, Wockhardt: Grant/Research Support
2179. Clinical Characteristics And Outcomes of Peritoneal Dialysis-Related Peritonitis Infection by Ampc β-lactamase-producing Enterobacterales
Authors: Navarro Alberto Ordinola , Salazar-Leal Jazmín Itzayana , Luis Bruno Ali Lopez 
Published: November 2023

Abstract

Background
Peritonitis is one of the most significant complications in patients undergoing peritoneal dialysis (PD). Even when appropriately treated, approximately 20% of all episodes are refractory to treatment. Enterobacteriaceae represent about 10% of cases of peritonitis associated with PD, gram-negative organisms that have inducible beta-lactamase genes known as AmpC are usually more severe clinically and are associated with worse outcomes, including an increased risk of hospitalization and death.

Methods
A retrospective case series in Centro Médico Nacional 20 de Noviembre in Mexico from 2020-2022. We included hospitalized patients diagnosed with peritonitis who met the following criteria: Cloudy peritoneal effluent, Abdominal pain, white-cell counts higher than 100/μL with at least 50% polymorphonuclear cells and culture of peritoneal effluent with the isolation of AmpC β-lactamase-producing Enterobacterales Identified by Vitek 2 and corroborated by phenotypic tests.

Results
30 episodes of peritonitis associated with PD by AmpC enterobacterales were identified. Clinical characteristics are summarized in Table1. Their resistance profiles are reported in Table2. Two patients had refractory peritonitis, 3 changed modality to hemodialysis, 2 had repeat peritonitis at 90 days due to Serratia marcescens and required removal. Four patients had a new episode of peritonitis due to another organism, and two died secondary to septic shock Table3. Notably, this infection may be related to poor hygiene conditions and poor techniques in replacing peritoneal fluid bags since we found that 71% of our patients acquired the infection at their homes. We found that 58% of patients treated with at least two antibiotics occupying 2 routes of administration (intravenous/intraperitoneal) had a good response.

Table 2
Table3

Conclusion
AmpC peritonitis can lead to refractoriness, loss of the catheter, change to hemodialysis and even death, which in our particular context, dual antibiotic therapy may be sufficient; however, removing the catheter improves the response to treatment and decreases refractory peritonitis. The relationship to complications by these agents are high (50% in this study), so aggressive management is necessary for better outcomes in case of isolation.

Disclosures
All Authors: No reported disclosures
2184. Clinical and Molecular Epidemiology of Carbapenem-Resistant Gram-Negative Bacilli in a Los Angeles Tertiary Medical Center
Authors: Sanchez John Michael , Contreras Deisy , Morgan Margie Ann 
Published: November 2023

Abstract

Background
Multidrug-resistant gram-negative bacilli are a significant public health threat, associated with 2.2 million deaths worldwide. Of particular importance is the rise of carbapenem-resistant gram-negative bacilli (CRGNB) given the limited options for treatment. The 30-day mortality associated with carbapenem-resistant Pseudomonas aeruginosa, carbapenem-resistant Enterobacterales, and carbapenem-resistant Acinetobacter baumannii ranges from 16 to 24%. In recognition of the substantial danger of these organisms, the World Health Organization has designated three CRGNB as Critical Priority pathogens.

Methods
Based on culture data and antimicrobial susceptibility testing, patients with CRGNB at Cedars-Sinai Medical Center from December 2022 to April 2023 were identified. Clinical data including patient demographics, prior carbapenem use, isolation of additional antibiotic-resistant organisms, subsequent antibiotic therapy, and mortality were compared among CRGNB.

Results
Of over 65 isolates, the largest proportion of isolates were cultured from pulmonary sources (44%), followed by urine (34%), skin (12%), blood (6%), and abdominal sources (4%). The most prevalent CRGNB isolated were Pseudomonas aeruginosa (65%), Escherichia coli (13%), and Klebsiella pneumoniae (7%). A majority of patients with CRGNB had prior documented carbapenem use (74%) and 71% of CRGNB were associated with at least one other antibiotic resistant organism. Cefiderocol was used to treat 12% of cases. However, 24% of CRGNB were not treated with antibiotics, often with the rationale that the isolate was likely a colonizing strain. The mortality rate among patients with CRGNB during our observation period was 18%.

Conclusion
CRGNB are emerging pathogens associated with prior carbapenem use and result in significant mortality. Subsets of organisms from our dataset will be further studied for relatedness using Fourier-Transform infrared spectroscopy and whole genome sequencing.

Disclosures
All Authors: No reported disclosures