bluuebunny/crossref_metadata_2025_split huggingface dataset:
1438. Dissecting the Multifaceted Nature of Antibiotic Resistance in Clinical Isolates of Neisseria gonorrhoeae by Natural Transformation
Authors: Huang Jianzhong , Ingraham Karen , Chan Pan , Rittenhouse Steve 
Published: January 2021

Abstract

Background
Neisseria gonorrhoeae (NG) causes the sexually transmitted disease gonorrhea. It has developed resistance to every antibiotic introduced for gonorrhea treatment such that NG clinical isolates with multidrug resistance (MDR) are increasingly common. We hypothesize that natural transformation could be used to transfer genetic determinants of antibiotic resistance from drug-resistant NG clinical isolates without pre-knowledge of the genetic determinants to a new background under antibiotic selection to generate isogenic transformants for further characterization.

Methods
Natural transformation, PCR amplification and DNA sequencing, and antibiotic susceptibility testing were used in the studies.

Results
We have validated the hypothesis using genomic DNA from an MDR including ciprofloxacin-resistant NG clinical isolate as a donor and a ciprofloxacin-susceptible NG isolate as a recipient under the selective pressure of ciprofloxacin. This led to a series of transformants that contain single or multiple genetic resistance determinants being generated depending on the resistance levels and transformation frequencies. Antibiotic susceptibility testing and genetic characterization of the transformants allowed us to (i) identify ciprofloxacin resistance determinants including efflux mutation mtrR-79 and target mutations GyrA S91F D95G and ParC D86N, (ii) quantify the contribution of each genetic determinant responsible for the ciprofloxacin resistance and (iii) regenerate the ciprofloxacin resistance phenotype of the donor isolate to detect multiple paths of possible resistance development for ciprofloxacin. Furthermore, we also validated the hypothesis with a novel antibiotic gepotidacin to identify pre-existing genetic determinants contributing to varying susceptibility to this antibiotic. Finally, we envision that, along with whole genome sequencing, natural transformation could be used to identify and quantify novel genetic resistance determinants to current or novel antibiotics in drug-resistant NG clinical isolates.

Conclusion
We demonstrated the utility of natural transformation in dissecting the multifaceted nature of antibiotic resistance in NG clinical isolates.

Disclosures
Jianzhong Huang, PhD, GlaxoSmithKline (Employee, Shareholder) Karen Ingraham, MS, GlaxoSmithKline (Employee, Shareholder) Pan Chan, PhD, GlaxoSmithKline (Employee, Shareholder) Steve Rittenhouse, PhD, GlaxoSmithKline (Employee, Shareholder)
117. Natural Language Processing: An Automated Alternative to Determining Inappropriate Group A Streptococcal Testing
Authors: Lee Brian R , Linafelter Alaina , Burns Alaina , Burris Allison , Jones Heather , Dusin Jarrod , El Feghaly Rana E 
Published: January 2021

Abstract

Background
Acute pharyngitis is one of the most common causes of pediatric health care visits, accounting for approximately 12 million ambulatory care visits each year. Rapid antigen detection tests (RADTs) for Group A Streptococcus (GAS) are one of the most commonly ordered tests in the ambulatory settings. Approximately 40–60% of RADTs are estimated to be inappropriate. Determination of inappropriate RADT frequently requires time-intensive chart reviews. The purpose of this study was to determine if natural language processing (NLP) can provide an accurate and automated alternative for assessing RADT inappropriateness.

Methods
Patients ≥ 3 years of age who received an RADT while evaluated in our EDs/UCCs between April 2018 and September 2018 were identified. A manual chart review was completed on a 10% random sample to determine the presence of sore throat or viral symptoms (i.e., conjunctivitis, rhinorrhea, cough, diarrhea, hoarse voice, and viral exanthema). Inappropriate RADT was defined as either absence of sore throat or reporting 2 or more viral symptoms. An NLP algorithm was developed independently to assign the presence/absence of symptoms and RADT inappropriateness. The NLP sensitivity/specificity was calculated using the manual chart review sample as the gold standard.

Results
Manual chart review was completed on 720 patients, of which 320 (44.4%) were considered to have an inappropriate RADT. When compared to the manual review, the NLP approach showed high sensitivity (se) and specificity (sp) when assigning inappropriateness (88.4% and 90.0%, respectively). Optimal sensitivity/specificity was also observed for select symptoms, including sore throat (se: 92.9%, sp: 92.5%), cough (se: 94.5%, sp: 96.5%), and rhinorrhea (se: 86.1%, sp: 95.3%). The prevalence of clinical symptoms was similar when running NLP on subsequent, independent validation sets. After validating the NLP algorithm, a long term monthly trend report was developed.
Figure Inappropriate GAS RADTs Determined by NLP, June 2018-May 2020

Conclusion
An NLP algorithm can accurately identify inappropriate RADT when compared to a gold standard. Manual chart review requires dozens of hours to complete. In contrast, NLP requires only a couple of minutes and offers the potential to calculate valid metrics that are easily scaled-up to help monitor comprehensive, long-term trends.

Disclosures
Brian R. Lee, MPH, PhD, Merck (Grant/Research Support)
1439. High Rates of Drug Resistance in Escherichia coli from a Pilot Antimicrobial Resistance Surveillance System in Cambodia
Authors: Nov Vandarith , Chau Darapheak , Chhea Chhorvann 
Published: January 2021

Abstract

Background
Antimicrobial resistance (AMR) is a major and growing global public health problem. The Cambodia Ministry of Health established a pilot laboratory-based AMR surveillance system for blood specimens in 2017. The objective of this study is to characterize AMR among pathogenic isolates from blood samples.

Methods
A retrospective analysis was performed using one year of data from a pilot AMR Surveillance system in Cambodia. Four blood culture isolate pathogens were included: Escherichia coli, Klebsiella pneumoniae, Salmonella Typhi /Salmonella Paratyphi A and Staphylococcus aureus. Blood culture isolates that were referred from eleven sentinel sites were analyzed at the National Public Health Laboratory for identification. Antibiotic susceptibility testing (AST) was done using disk diffusion, minimum inhibitory concentration method following Clinical Laboratory Standard Institute (CLSI) guidelines.

Results
Among 214 pathogenic isolates from blood samples, E.coli was the most common (56.1%), followed by Salmonella Typhi/Salmonella Paratyphi A (18.7%), Staphylococcus aureus (13.5%), and Klebsiella pneumoniae (11.7%). Methicillin Resistance Staphylococcus aureus (MRSA) was detected in half of the isolates. E.coli was resistant to ampicillin (94.4%), trimethoprim-sulfamethoxazole (84.5%), and ceftriaxone (79.2%). Salmonella Typhi was resistant to ampicillin (73.3%) and trimethoprim-sulfamethoxazole (60.0%) and Salmonella Paratyphi A were resistant to fluoroquinolones (91.7%). For last resort antibiotics, E.coli was resistant to carbapenem groups (3.2% for imipenem, 4.9% for meropenem, and 5.0% for ertapenem). Klebsiella pneumoniae was not resistant to any groups.

Conclusion
E.coli was found at high rates in blood samples and was resistant to common antibiotics used in Cambodia. These pilot data show the importance of establishing a national AMR surveillance system in Cambodia to monitor AMR trends following GLASS guidelines.

Disclosures
All Authors: No reported disclosures
1442. Acquisition and Transferability Mechanisms of Mercury Resistance Genes in Latin-American Staphylococcus aureus Strains
Authors: Ortega-Recalde Oscar , Echeverri Aura M , Rios Rafael , Carvajal Lina , Hernandez Angie K , Espitia-Acero Catalina , Solano-Gutierrez Sebastian , Shropshire William C , Arias Cesar A , Diaz Lorena , Rincon Sandra , Reyes Jinnethe 
Published: January 2021

Abstract

Background
Latin-American (LA) countries are among the largest mercury (Hg) polluters in the world. Fittingly, a significant high frequency (&gt;50%) of Hg resistance genes (MRG) has been observed in LA MRSA genomes, including USA300-LV clone, which contains the genomic element COMER, encoding for copper and Hg resistance genes adjacent to SCCmecIVc/E. Co-selection of MRG and antibiotic resistance genes may be facilitated by shared transferable genetic elements, nevertheless, analyses of the genetic MRG context in strains other than USA300-LV are lacking. In this study, we aimed to characterize possible mechanisms of acquisition and transfer of MRG in LA S. aureus.

Methods
We sequenced 6 MRSA and 2 MSSA clinical isolates harboring MRG from Colombia, Ecuador, Peru and Chile using short-read (Illumina) and long-read (ONT) sequencing. Hybrid assemblies were constructed using Flye and iterative polishing with Medaka and Racon. Identification of insertion sequences, rearrangements and assessment of the genomic context was investigated using ISfinder, MAUVE, PlasmidFinder and SnapGene.

Results
Highly contiguous genome assemblies allowed us to identify the localization and genetic background of MRG. For MRSA belonging to USA300-LV (SCCmecIVc/E) and Brazilian (SCCmecIII) clones, we confirmed the presence of MRG within SCCmec. In contrast, for the 4 MRSA belonging to Chilean/Cordobes clone (SCCmecI), collected from Colombia, Chile and Peru, MRG were located on ~30kbp plasmids genetically related that also contained the blaZ beta-lactamase and cadmium/arsenic resistance genes. In MSSA strains, we observed both plasmidic and chromosomal localizations of MGR. Interestingly, in one of the MSSA, MRG were inserted downstream of orfX, along with repA, suggesting a plasmidic origin. In all these cases, MRG were flanked by IS6 family elements.

Conclusion
Genomic architecture of SCCmec types IVc/E and III might facilitate MRG transferability, whereas for the highly prevalent Chilean/Cordobes clone (SCCmecI) MRG acquisition occurs through plasmids. Our findings underscore the mechanisms of MRG transference in LA S. aureus likely related to antibiotic resistance co-selection.

Disclosures
Cesar A. Arias, MD, MSc, PhD, FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)
1444. Characterization of a Novel Pathogen in Immunocompromised Patients: Elizabethkingia Anopheles
Authors: Yasmin Mohamad , Marshall Steven , Marshall Emma , Cmolik Anna , Boucher Helen , Vila Alejandro , Bonomo Robert A 
Published: January 2021

Abstract

Background
A 60-year-old woman with acute myeloid leukemia developed fever and cough post induction chemotherapy. Meropenem and vancomycin were administered as empiric therapy. A bloodstream infection due to resistant E. meningoseptica was initially identified. Ceftazidime-avibactam and aztreonam (ATM) were next administered. Minocycline and trimethoprim/sulfamethoxazole (TMP/SMX) were added as the patient deteriorated and died ten days later.

Methods
MIC testing /disc diffusion assays were done according to CLSI guidelines. Whole genome sequencing (WGS) was performed, assembled, and annotated using PATRIC.org and compared to Institute Pasteur cgMLST instance of the BIGSdb database tool. Resistant genes and plasmids were identified by the Center for Genomic epidemiology.

Results
Core genome MLST and average nucleotide identity of 99.46% revealed the organism as E. anophelis, not E. meningoseptica. The WGS assembly resulted in 118 contigs with a chromosome of 4,110,726 bp. Plasmids were not identified. Resistance genes blaB11, blaGOB-13 (a metallo beta-lactamase) and blaCME-1 were detected. E. anophelis was resistant to multiple drug classes including aminoglycosides, vancomycin, cephalosporins, and carbapenems. Mechanism based susceptibility testing using double-disc diffusion assays demonstrated susceptibility to cefiderocol (MIC 1 µg/ml), minocycline, TMP/SMX, linezolid, and modestly wider zones of inhibition with the addition of ATM. Susceptibility results are shown in Table 1.
Antimicrobial susceptibility testing using various BLIs and combinations with Aztreonam

Conclusion
This is one of the first reports describing E. anophelis in immunocompromised hosts. More thorough testing methods are needed for distinguishing between species in the Elizabethkingia genus in hospital settings as there is concern that this may represent a novel opportunistic infection. Per CDC, E. anophelis outbreaks have occurred in three Midwestern states in 2016. Vigilance is warranted to the role of this pathogen in immunocompromised hosts.

Disclosures
Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)
1445. Deciphering the Role of the Y221H Ω-loop Substitution in Pseudomonas-derived Cephalosporinase (PDC) in Cephalosporin Resistance
Authors: Mack Andrew R , Barnes Melissa D , Taracila Magdalena A , Mojica Maria F , Kumar Vijay , Rutter Joseph , van den Akker Focco , Page Malcolm , Haider Shozeb , Bonomo Robert A 
Published: January 2021

Abstract

Background
Antimicrobial resistance is a major global health threat. Pseudomonas aeruginosa is a leading cause of nosocomial infections and a key opportunistic pathogen in cystic fibrosis. Multidrug resistant strains are classified as a “serious threat” by the CDC. Pseudomonas-derived cephalosporinase (PDC) is largely responsible for β-lactam antibiotic resistance in P. aeruginosa. Single amino acid substitutions in the essential Ω-loop region (e.g. Y221H by structural alignment-based numbering of class C β-lactamases) have been shown to enhance hydrolysis of ceftazidime (CAZ) and ceftolozane (TOL), limiting therapeutic options for P. aeruginosa.

Methods
We undertook detailed studies to explore the mechanisms by which Y221H enhances CAZ and TOL MICs. MIC measurements were performed per CLSI guidelines using MH Agar. Thermal stability was determined by circular dichroism. Enzyme kinetic properties were determined using spectrophotometric techniques. Molecular dynamics techniques were used to predict structural changes.

Results
E. coli expressing blaPDC-3-Y221H is less susceptible to CAZ (MIC 0.5 mg/L WT → 8 mg/L Y221H) and TOL (MIC 2 mg/L WT → 16 mg/L Y221H). Using steady-state kinetic analysis, Y221H was found to hydrolyze CAZ with a KM = 585 µM, a kcat = 3.4 sec-1, and kcat/KM = 0.0058 µM-1s-1. With cephalothin, a good PDC substrate, we observed KM = 26.6 µM, kcat = 70.1 s-1, and kcat/KM = 2.6 µM-1 s-1 for Y221H. Using Electrospray ionization mass spectrometry (ESI-MS), CAZ was detected covalently bound to WT, but not Y221H when incubated at 1000-fold molar excess. Avibactam (AVI) inhibited Y223H (Ki = 70 nM vs. 19 nM for WT). Y221H thermal stability decreased by 5°C (Tm = 47°C vs 52°C WT). AVI at 10-fold molar excess does not increase Tm in Y221H or WT. WT-MetaDynamics (WT MDS) predicts the opening of a hidden pocket by repositioning residue 221 (Figure 1).).
Figure 1: (Left) We carried out enhanced sampling metadynamics simulations to generate free-energy landscapes as a function of the dihedral angles of residue 221. This identifies the differences in the dynamics of the tyrosyl side chains in the wild type Y221 and the imidazole ring of the H221 variant. (Right) The rotation of the side chain in H221 opens a cryptic pocket (green mesh), which is occluded in the wild type. The Ω-loop is colored red.

Conclusion
PDC-3 Y221H increases CAZ & TOL MICs and alters catalytic activity, primarily by a change in kcat. Our modelling analyses suggest altered conformational flexibility and structure-function relationships in the Ω-loop. These results help to advance our understanding of PDC and will inform development of novel antibiotics and inhibitors.

Disclosures
Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)
1448. Forgiveness of BIC/FTC/TAF: In Vitro Simulations of Intermittent Poor Adherence Find Limited HIV-1 Breakthrough and High Barrier to Resistance
Authors: Mulato Andrew , Acosta Rima K , Yant Stephen R , Cihlar Tomas , White Kirsten L 
Published: January 2021

Abstract

Background
Short lapses in adherence to ARVs can lead to virologic failure and emergence of resistance. Previous in vitro studies of regimen “forgiveness” simulated drug exposures of perfect adherence or short-term suboptimal adherence with bictegravir+emtricitabine+tenofovir alafenamide (BIC+FTC+TAF) and with dolutegravir and lamivudine (DTG+3TC). Here, viral breakthrough (VB) and resistance development were evaluated under alternating high and low drug exposures simulating variable adherence levels.

Methods
Wild-type HIV-1 (IIIb)-infected MT-2 cells were exposed to drug combinations and monitored for VB. Experiments alternated between high and low drug concentrations of either BIC+FTC+TAF or DTG+3TC (Table 1). Drug concentrations for each regimen were determined using human plasma-free adjusted clinical trough concentrations (Cmin), at simulated Cmin after missing 2 or 4 consecutive doses (Cmin-2 and Cmin-4) based on drug half-lives. Emergent HIV-1 were genotyped by deep sequencing and a 2% threshold.

Results
In these experiments, constant drug concentrations corresponding to full adherence (Cmin) did not lead to VB. Using Cmin concentrations for one week followed by constant Cmin-2 exposures for 4 weeks, DTG+3TC had VB and emergence of M184V/I in reverse transcriptase (RT) but there was no VB for BIC+FTC+TAF. Using alternating drug exposures of Cmin (weeks 1 and 3) and Cmin-2 or Cmin -4 (weeks 2, 4, and 5), VB was not observed with BIC+FTC+TAF, and VB was decreased or delayed with DTG+3TC compared to DTG+3TC held at Cmin-2 or Cmin-4. Resistance development was observed in some cultures with VB: 1 culture with BIC+FTC+TAF had G163R in IN and 19 cultures with DTG+3TC had INSTI and RT resistance including 10 with M184V/I.
Table 1. Summary of Breakthrough Frequency and Resistance Development

Conclusion
BIC+FTC+TAF has high in vitro forgiveness and consistent protection against emergence of drug resistance during simulations of short lapses in adherence. Higher DTG+3TC exposure, whether constant or intermittent, was better at preventing or delaying VB than lower DTG+3TC exposures, but DTG+3TC was less forgiving than BIC+FTC+TAF. Prevention of viral replication and resistance development is necessary to maintain lifelong viral suppression, particularly in the real world where drug adherence is often imperfect.

Disclosures
Andrew Mulato, BS, MBA, Gilead Sciences, Inc. (Employee, Shareholder) Rima K. Acosta, BS, Gilead Sciences, Inc. (Employee, Shareholder) Stephen R. Yant, PhD, Gilead Sciences, Inc. (Employee, Shareholder) Tomas Cihlar, PhD, Gilead Sciences, Inc. (Employee, Shareholder) Kirsten L. White, PhD, Gilead Sciences, Inc. (Employee, Shareholder)
118. Optimizing the management of coagulase-negative staphylococci (CoNS) contaminants by reporting the species name
Authors: Stone Tyler J , Palavecino Elizabeth , Palavecino Elizabeth , Jessica Smith , Beardsley James , Johnson James , Luther Vera , Ohl Christopher , Ohl Christopher , Williamson John 
Published: January 2021

Abstract

Background
CoNS are common isolates in blood cultures (BCx), but many are contaminants contributing to unnecessary antibiotic use. When CoNS are isolated from multiple BCx, different species and/or different susceptibility patterns may suggest contamination. Species reporting of CoNS is not performed at all hospitals. The purpose of this study was to characterize antibiotic use attributable to CoNS positive (pos) BCx and determine if reporting CoNS species could help reduce unnecessary antibiotics.

Methods
A retrospective chart review was conducted of inpatients at an academic medical center before (Jan-June 2017) and after (Sept 2019-Feb 2020) implementation of CoNS species reporting. CoNS species were hidden to providers in the before group. Patients (pts) ≥18 years old with ≥1 BCx pos for CoNS were included. Pts who were neutropenic, treated with anti-staphylococcal antibiotics (SAbx) for a non-CoNS infection, or treated for CoNS with non-SAbx were excluded. Pts were categorized by number of pos BCx (1 vs ≥2). In each period, a random sample of pts was screened until 50 pts with 1 CoNS pos BCx were included. Additional data were collected until at least 50 pts with ≥2 pos BCx were included in each period. The primary outcome was use of SAbx among pts in each group before and after species reporting. Additional analyses were performed to compare the use of SAbx among subsets with same/different species and/or susceptibilities.

Results
203 pts were included, 102 before and 101 after. 51% and 50% had ≥2 pos BCx in the before and after groups, respectively. S. epidermidis was isolated more frequently in pts with ≥2 pos BCx (75% vs 50%, p&lt; 0.001). 77% of pts received at least 1 SAbx (97% vancomycin). Median SAbx days of therapy per pt (DOTs) was greater among pts with ≥2 pos BCx (1 vs 5, p&lt; 0.001). There was no difference in overall DOTs between the two periods (3 vs 2, p=0.25). However, among pts with ≥2 pos BCx, median DOTs was less in the after period (6.5 vs 3, p=0.016). Among pts with 1 pos BCx, median DOTs was 1 in both periods.
Median Anti-Staphylococcal Antibiotic Days of Therapy per Patient (≥ 2 positive cultures)

Conclusion
CoNS species reporting was associated with decreased SAbx use for pts with ≥2 pos BCx, suggesting that knowing the species helps in determining likelihood of true infection. Institutions may realize improved stewardship metrics of SAbx by implementing CoNS species reporting for pos BCx.

Disclosures
Tyler J. Stone, PharmD, Paratek (Research Grant or Support) Elizabeth Palavecino, MD, Paratek (Grant/Research Support)Paratek (Grant/Research Support) John Williamson, PharmD, Paratek (Research Grant or Support)