scholarly journals 707. Clarifying the Role of CrrB in Polymxyin-resistant Klebsiella pneumoniae Clinical Isolates Utilizing a Novel CRISPR-Cas9 System

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S254-S255
Author(s):  
Thomas McConville ◽  
Marla Giddins ◽  
Nenad Macesic ◽  
Anne-Catrin Uhlemann
Keyword(s):  
Genetic Manipulation ◽  
Alternative Pathway ◽  
Negative Regulator ◽  
Missense Mutations ◽  
Downstream Targets ◽  
Qrt Pcr ◽  
Carbapenem Resistant ◽  
The Impact ◽  
Carbapenem Resistant Enterobacteriaceae

Abstract Background Polymyxin resistance (PR) threatens the mainstay of therapy for carbapenem-resistant Enterobacteriaceae (CRE) infections. While mgrB disruption accounts for most cases of PR, missense mutations in crrB have been proposed as an alternative pathway for PR through PmrA/B/C upregulation of the pmrHFIJKLM operon. It remains unknown if CrrB acts as a positive or negative regulator on its downstream targets. Methods We assembled a CRISPR-Cas9 system for gene knockouts (KO) in CRE K. pneumoniae (CRKP) using zeocin as a selectable marker. We chose a polymyxin susceptible (PS) and a PR isolate with a missense mutation in crrB (L87V) (NR5337 and NR5083, respectively) for KO. Isolates were transformed with a crrB KO plasmid, grown with zeocin selection, induced with arabinose, and plated on low-salt LB-zeocin/arabinose. KOs were confirmed via PCR and Sanger sequencing. Polymyxin susceptibility was performed with broth-microdilution. Gene expression was determined by qRT-PCR of cDNA extracts. Results Colistin MIC following crrB KO of NR5337 (PS) remained unchanged. In contrast, crrB KO of NR5083 (PR), decreased polymyxin MIC (MIC >128 to 1.0 μg/mL). qRT-PCR of NR5083 did not show increased expression of pmrA/C, nor pmrK. NR5083 ^crrB showed a small decrease in phoQ expression, compared with NR5083, but similar expression of phoP, pmrA/C and pmrK (Table 1). Conclusion Polymyxin MIC decreased >128 fold after crrB KO in a PR isolate, but colistin MIC remained unchanged after KO in a PS isolate. CrrB mutations in PR isolates may confer a gain of function with CrrB acting as a positive regulator on its downstream targets. Contrary to previous literature, no upregulation of pmrA/C and pmrHFIJKLM was detected. Differences in crrB mutations or clonal background may explain this finding. CRISPR-Cas9 may serve as a reliable system for genetic manipulation of CRKP. Further data on the impact of individual crrB missense mutations are needed. Disclosures A. C. Uhlemann, Merck: Investigator, Grant recipient.

A National Intervention to Prevent the Spread of Carbapenem-Resistant Enterobacteriaceae in Israeli Post-Acute Care Hospitals

2014 ◽  
Vol 35 (7) ◽  
pp. 802-809 ◽  
Author(s):  
Debby Ben-David ◽  
Samira Masarwa ◽  
Amos Adler ◽  
Hagit Mishali ◽  
Yehuda Carmeli ◽  
...  
Keyword(s):  
Infection Control ◽  
Acute Care ◽  
Acute Care Hospitals ◽  
Control Measures ◽  
Resistant Bacteria ◽  
Carbapenem Resistant ◽  
Control Intervention ◽  
The Impact ◽  
Carbapenem Resistant Enterobacteriaceae ◽  
Post Acute Care

ObjectivePatients hospitalized in post-acute care hospitals (PACHs) constitute an important reservoir of antimicrobial-resistant bacteria. High carriage prevalence of carbapenem-resistant Enterobacteriaceae (CRE) has been observed among patients hospitalized in PACHs. The objective of the study is to describe the impact of a national infection control intervention on the prevalence of CRE in PACHs.DesignA prospective cohort interventional study.SettingThirteen PACHs in Israel.InterventionA multifaceted intervention was initiated between 2008 and 2011 as part of a national program involving all Israeli healthcare facilities. The intervention has included (1) periodic on-site assessments of infection control policies and resources, using a score comprised of 16 elements; (2) assessment of risk factors for CRE colonization; (3) development of national guidelines for CRE control in PACHs involving active surveillance and contact isolation of carriers; and (4) 3 cross-sectional surveys of rectal carriage of CRE that were conducted in representative wards.ResultsThe infection control score increased from 6.8 to 14.0 (P < .001) over the course of the study period. A total of 3,516 patients were screened in the 3 surveys. Prevalence of carriage among those not known to be carriers decreased from 12.1% to 7.9% (P = .008). Overall carrier prevalence decreased from 16.8% to 12.5% (P = .013). Availability of alcohol-based hand rub, appropriate use of gloves, and a policy of CRE surveillance at admission to the hospital were independently associated with lower new carrier prevalence.ConclusionA nationwide infection control intervention was associated with enhanced infection control measures and a reduction in the prevalence of CRE in PACHs.

scholarly journals Role of unfolded proteins in lung disease

Thorax ◽  
2020 ◽  
Vol 76 (1) ◽  
pp. 92-99
Author(s):  
Kirsty L Bradley ◽  
Clare A Stokes ◽  
Stefan J Marciniak ◽  
Lisa C Parker ◽  
Alison M Condliffe
Keyword(s):  
Protein Synthesis ◽  
Er Stress ◽  
Respiratory Diseases ◽  
Viral Infections ◽  
Genetic Manipulation ◽  
Experimental Models ◽  
Misfolded Proteins ◽  
Pharmacological Agents ◽  
The Impact

The lungs are exposed to a range of environmental toxins (including cigarette smoke, air pollution, asbestos) and pathogens (bacterial, viral and fungal), and most respiratory diseases are associated with local or systemic hypoxia. All of these adverse factors can trigger endoplasmic reticulum (ER) stress. The ER is a key intracellular site for synthesis of secretory and membrane proteins, regulating their folding, assembly into complexes, transport and degradation. Accumulation of misfolded proteins within the lumen results in ER stress, which activates the unfolded protein response (UPR). Effectors of the UPR temporarily reduce protein synthesis, while enhancing degradation of misfolded proteins and increasing the folding capacity of the ER. If successful, homeostasis is restored and protein synthesis resumes, but if ER stress persists, cell death pathways are activated. ER stress and the resulting UPR occur in a range of pulmonary insults and the outcome plays an important role in many respiratory diseases. The UPR is triggered in the airway of patients with several respiratory diseases and in corresponding experimental models. ER stress has been implicated in the initiation and progression of pulmonary fibrosis, and evidence is accumulating suggesting that ER stress occurs in obstructive lung diseases (particularly in asthma), in pulmonary infections (some viral infections and in the setting of the cystic fibrosis airway) and in lung cancer. While a number of small molecule inhibitors have been used to interrogate the role of the UPR in disease models, many of these tools have complex and off-target effects, hence additional evidence (eg, from genetic manipulation) may be required to support conclusions based on the impact of such pharmacological agents. Aberrant activation of the UPR may be linked to disease pathogenesis and progression, but at present, our understanding of the context-specific and disease-specific mechanisms linking these processes is incomplete. Despite this, the ability of the UPR to defend against ER stress and influence a range of respiratory diseases is becoming increasingly evident, and the UPR is therefore attracting attention as a prospective target for therapeutic intervention strategies.

scholarly journals Water as a Source of Antimicrobial Resistance and Healthcare-Associated Infections

Pathogens ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 667
Author(s):  
Claire Hayward ◽  
Kirstin E. Ross ◽  
Melissa H. Brown ◽  
Harriet Whiley
Keyword(s):  
Antimicrobial Resistance ◽  
Hot Spot ◽  
Developed Countries ◽  
Healthcare Associated Infections ◽  
Carbapenem Resistant ◽  
The Us ◽  
Waterborne Infection ◽  
Healthcare Associated ◽  
Carbapenem Resistant Enterobacteriaceae

Healthcare-associated infections (HAIs) are one of the most common patient complications, affecting 7% of patients in developed countries each year. The rise of antimicrobial resistant (AMR) bacteria has been identified as one of the biggest global health challenges, resulting in an estimated 23,000 deaths in the US annually. Environmental reservoirs for AMR bacteria such as bed rails, light switches and doorknobs have been identified in the past and addressed with infection prevention guidelines. However, water and water-related devices are often overlooked as potential sources of HAI outbreaks. This systematic review examines the role of water and water-related devices in the transmission of AMR bacteria responsible for HAIs, discussing common waterborne devices, pathogens, and surveillance strategies. AMR strains of previously described waterborne pathogens including Pseudomonas aeruginosa, Mycobacterium spp., and Legionella spp. were commonly isolated. However, methicillin-resistant Staphylococcus aureus and carbapenem-resistant Enterobacteriaceae that are not typically associated with water were also isolated. Biofilms were identified as a hot spot for the dissemination of genes responsible for survival functions. A limitation identified was a lack of consistency between environmental screening scope, isolation methodology, and antimicrobial resistance characterization. Broad universal environmental surveillance guidelines must be developed and adopted to monitor AMR pathogens, allowing prediction of future threats before waterborne infection outbreaks occur.

scholarly journals The conformational and mutational landscape of the ubiquitin-like marker for the autophagosome formation in cancer

2019 ◽  
Author(s):  
Burcu Aykac Fas ◽  
Mukesh Kumar ◽  
Valentina Sora ◽  
Maliha Mashkoor ◽  
Matteo Lambrughi ◽  
...  
Keyword(s):  
Physical Models ◽  
Missense Mutations ◽  
Cancer Types ◽  
Neutral Mutations ◽  
And Function ◽  
Cellular Components ◽  
The Impact ◽  
Pan Cancer ◽  
Structural Ensemble

AbstractAutophagy is a cellular process to recycle damaged cellular components and its modulation can be exploited for disease treatments. A key autophagy player is a ubiquitin-like protein, LC3B. Compelling evidence attests the role of autophagy and LC3B in different cancer types. Many LC3B structures have been solved, but a comprehensive study, including dynamics, has not been yet undertaken. To address this knowledge gap, we assessed ten physical models for molecular dynamics for their capabilities to describe the structural ensemble of LC3B in solution using different metrics and comparison with NMR data. With the resulting LC3B ensembles, we characterized the impact of 26 missense mutations from Pan-Cancer studies with different approaches. Our findings shed light on driver or neutral mutations in LC3B, providing an atlas of its modifications in cancer. Our framework could be used to assess the pathogenicity of mutations by accounting for the different aspects of protein structure and function altered by mutational events.

scholarly journals Nosocomial Pathogens: An In-Depth Analysis of the Vectorial Potential of Cockroaches

2019 ◽  
Vol 4 (1) ◽  
pp. 14 ◽  
Author(s):  
Eric S. Donkor
Keyword(s):  
Nosocomial Infections ◽  
Social Consequences ◽  
Antibiotic Resistant ◽  
Nosocomial Pathogens ◽  
Healthcare Facilities ◽  
Carbapenem Resistant ◽  
Depth Analysis ◽  
Healthcare Associated ◽  
Carbapenem Resistant Enterobacteriaceae

Nosocomial or healthcare-associated infections are regarded as the most frequent adverse event that threatens patients’ safety and has serious economic and social consequences. Cockroach infestation is common in many hospitals, especially in the developing world. Common nosocomial pathogens isolated from cockroaches include Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae. Cockroaches also harbor epidemiologically significant antibiotic-resistant organisms, such as carbapenem-resistant Enterobacteriaceae, which complicate nosocomial infections. Therefore, cockroaches constitute an important vector for nosocomial pathogens, and there should be zero tolerance for their presence in healthcare facilities. This paper aims to elucidate the possible role of cockroaches in nosocomial infections by reviewing the relevant research publications.

scholarly journals 2130. Detection of Carbapenemase-Producing Organisms and Impact on Antimicrobial Utilization for Carbapenem-Resistant Enterobacteriaceae (CRE) Infections

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S721-S722
Author(s):  
Sarah Brooks Minor ◽  
Jose Alexander
Keyword(s):  
Average Duration ◽  
Screening Method ◽  
Carbapenem Resistant ◽  
Antimicrobial Utilization ◽  
Carbapenemase Gene ◽  
Testing Algorithm ◽  
The Impact ◽  
Accurate Reporting ◽  
Carbapenem Resistant Enterobacteriaceae ◽  
Selection Of

Abstract Background CREs are feared pathogens with resistance occurring through the production of carbapenemases. Identification of carbapenemase-producing (CP) organisms assists with proper antimicrobial selection of commonly used agents, such as ceftazidime/avibactam (CA), meropenem/vaborbactam (MV), and tigecycline (TG). AdventHealth Orlando implemented a CRE screening method based on meropenem (MER) and a confirmatory CRE PCR testing in March 2018. Prior to implementing this test, patients were deemed to have CRE infections (CREI) if the organism demonstrated resistance to any carbapenem. The objective of this study was to evaluate the impact of this testing on the utilization of anti-CRE antibiotics. Methods This was a retrospective pre (March 2017–February 2018)- and post (March 2018–February 2019)-implementation study examining the impact of CRE PCR testing. Outcomes included the number of antibiotic days saved, average duration of therapy (DOT), median length of stay (LOS), and change in CP-CRE prevalence. The intervention consisted of the implementation of CRE PCR testing and included inpatients > 18 years old who received either CA, MV, or TG for the treatment of a CREI. Results Post-implementation, 30 unique patients were identified as having a positive K. pneumoniae carbapenemase gene by PCR, indicating a CP-CRE and received CA, MV, or TG; whereas, 42 patients in the pre-implementation group had a CREI and received CA, MV, or TG. Testing to identify CP-CREs led to a 50% reduction in the number of antibiotic days for CA, MV, and TG (575 vs. 287 days, P < 0.0001). Additionally, the average DOT decreased by 2.5 days in the post-implementation group (10.5 days vs. 8 days, P = 0.18) along with a 3.5-day shorter median LOS (15 days vs. 11.5 days, P = 0.48). The CRE prevalence based on resistance only to MER is 0.27%, compared with the previously reported rate of 2.5% that included resistance to ertapenem or MER. Conclusion Implementing CRE PCR testing to identify CP-CRE organisms resulted in a significant reduction in utilization of anti-CRE agents for CREIs. Additionally, the testing algorithm allowed for accurate reporting of our local CRE prevalence. By avoiding CA, MV, or TG in patients without CP-CREs, this has the potential to optimize therapy while reducing collateral damage associated with broad-spectrum agents. Disclosures All authors: No reported disclosures.

scholarly journals eVIP2: Expression-based variant impact phenotyping to predict the function of gene variants

2021 ◽  
Vol 17 (7) ◽  
pp. e1009132
Author(s):  
Alexis M. Thornton ◽  
Lishan Fang ◽  
April Lo ◽  
Maria McSharry ◽  
David Haan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colon Adenocarcinoma ◽  
Mapk Signaling ◽  
Negative Regulator ◽  
Gene Variants ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Functional Impact ◽  
Pathways Analysis ◽  
The Impact

While advancements in genome sequencing have identified millions of somatic mutations in cancer, their functional impact is poorly understood. We previously developed the expression-based variant impact phenotyping (eVIP) method to use gene expression data to characterize the function of gene variants. The eVIP method uses a decision tree-based algorithm to predict the functional impact of somatic variants by comparing gene expression signatures induced by introduction of wild-type (WT) versus mutant cDNAs in cell lines. The method distinguishes between variants that are gain-of-function, loss-of-function, change-of-function, or neutral. We present eVIP2, software that allows for pathway analysis (eVIP Pathways) and usage with RNA-seq data. To demonstrate the eVIP2 software and approach, we characterized two recurrent frameshift variants in RNF43, a negative regulator of Wnt signaling, frequently mutated in colorectal, gastric, and endometrial cancer. RNF43 WT, RNF43 R117fs, RNF43 G659fs, or GFP control cDNA were overexpressed in HEK293T cells. Analysis with eVIP2 predicted that the frameshift at position 117 was a loss-of-function mutation, as expected. The second frameshift at position 659 has been previously described as a passenger mutation that maintains the RNF43 WT function as a negative regulator of Wnt. Surprisingly, eVIP2 predicted G659fs to be a change-of-function mutation. Additional eVIP Pathways analysis of RNF43 G659fs predicted 10 pathways to be significantly altered, including TNF-α via NFκB signaling, KRAS signaling, and hypoxia, highlighting the benefit of a more comprehensive approach when determining the impact of gene variant function. To validate these predictions, we performed reporter assays and found that each pathway activated by expression of RNF43 G659fs, but not expression of RNF43 WT, was identified as impacted by eVIP2, supporting that RNF43 G659fs is a change-of-function mutation and its effect on the identified pathways. Pathway activation was further validated by Western blot analysis. Lastly, we show primary colon adenocarcinoma patient samples with R117fs and G659fs variants have transcriptional profiles similar to BRAF missense mutations with activated RAS/MAPK signaling, consistent with KRAS signaling pathways being GOF in both variants. The eVIP2 method is an important step towards overcoming the current challenge of variant interpretation in the implementation of precision medicine. eVIP2 is available at https://github.com/BrooksLabUCSC/eVIP2.

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