bluuebunny/crossref_metadata_2025_split huggingface dataset:
A multicenter performance evaluation of cefiderocol MIC results: ComASP in comparison to CLSI broth microdilution
Authors: Koeth L. M. , DiFranco-Fisher J. M. , Palavecino E. , Kilic A. , Hardy D. , Vicino D. , Stracquadanio S. , Stefani S. 
Published: December 2024

ABSTRACT

The performance of the Liofilchem Compact Antimicrobial Susceptibility Panel (ComASP) Cefiderocol was evaluated in a multicenter study. Enterobacterales,
Acinetobacter baumannii
, and
Pseudomonas aeruginosa
clinical isolates and challenge isolates were tested by three and one sites, respectively. Minimum inhibitory concentration (MIC) testing was performed by the Clinical and Laboratory Standards Institute (CLSI) broth microdilution and ComASP, which included two reading endpoints (CLSI read; MIC is the first well in which reduction of growth is <1 mm or light haze/faint turbidity] and ComASP [ComASP read; MIC is the first well at which 100% inhibition of growth occurs]). Each site performed reproducibility and quality control (QC) by ComASP and broth microdilution (BMD). Reproducibility was excellent (97.4% within ±1 dilution of modal MIC). All QC results were within CLSI QC ranges by BMD and ComASP, except for two
E. coli
ATCC 25922 results from one site. Essential agreement for combined clinical and challenge Enterobacterales was 84.3% (CLSI read) and 95.7% (ComASP read),
P. aeruginosa
was 83.3% (CLSI read) and 93.7% (ComASP read), and
A. baumannii
was 78.3% (CLSI read) and 96.7% (ComASP read). Categorical agreement for Enterobacterales was 92.4% for both CLSI read and ComASP read, for
P. aeruginosa
was 89.7% (CLSI read) and 92.1% (ComASP read), and for
A. baumannii
was 72.8% (CLSI read) and 91.3% (ComASP read). There were no very major errors using the ComASP read. One very major error for
P. aeruginosa
occurred using the CLSI read method. Three very major errors for
A. baumannii
occurred using the CLSI read method. ComASP Cefiderocol was shown to be a reliable method for testing cefiderocol MIC against relevant clinical isolates when ComASP read is used.

IMPORTANCE
There are very limited commercial methods available to clinical laboratories for cefiderocol minimum inhibitory concentration (MIC) testing. The Compact Antimicrobial Susceptibility Panel (ComASP) Cefiderocol method includes iron-depleted cation-adjusted Mueller–Hinton broth, which eliminates variability in cefiderocol MIC results based on iron levels. The lyophilized multi-well format of ComASP also provides for room temperature storage. In comparison to what an individual lab may do for method verification, this multi-site, multi-isolate study provides a robust evaluation and greater assurance to clinical microbiologists of the method's accurate and reproducible performance.
High-sensitivity detection of
Mycobacterium tuberculosis
DNA in tongue swab samples
Authors: Olson Alaina M. , Wood Rachel C. , Weigel Kris M. , Yan Alexander J. , Lochner Katherine A. , Dragovich Rane B. , Luabeya Angelique K. , Yager Paul , Hatherill Mark , Cangelosi Gerard A. 
Published: December 2024

ABSTRACT

Tongue swab (TS) sampling combined with quantitative PCR (qPCR) to detect
Mycobacterium tuberculosis
(MTB) DNA is a promising alternative to sputum testing for tuberculosis (TB) diagnosis. In prior studies, the sensitivity of tongue swabbing has usually been lower than sputum. In this study, we evaluated two strategies to improve sensitivity. In one, centrifugation was used to concentrate tongue dorsum bacteria from 2-mL suspensions eluted from high-capacity foam swab samples. The pellets were resuspended as 500-µL suspensions, and then mechanically lysed prior to dual-target qPCR to detect MTB insertion elements IS
6110
and IS
1081
. Fractionation experiments demonstrated that most of the MTB DNA signal in clinical swab samples (99.22% ± 1.46%) was present in the sedimentable fraction. When applied to archived foam swabs collected from 124 South Africans with presumptive TB, this strategy exhibited 83% sensitivity (71/86) and 100% specificity (38/38) relative to sputum microbiological reference standard (MRS; sputum culture and/or Xpert Ultra). The second strategy used sequence-specific magnetic capture (SSMaC) to concentrate DNA released from MTB cells. This protocol was evaluated on archived Copan FLOQSwabs flocked swab samples collected from 128 South African participants with presumptive TB. Material eluted into 500 µL buffer was mechanically lysed. The suspensions were digested by proteinase K, hybridized to biotinylated dual-target oligonucleotide probes, and then concentrated ~20-fold using magnetic separation. Upon dual-target qPCR testing of concentrates, this strategy exhibited 90% sensitivity (83/92) and 97% specificity (35/36) relative to sputum MRS. These results point the way toward automatable, high-sensitivity methods for detecting MTB DNA in TS.

IMPORTANCE
Improved testing for tuberculosis (TB) is needed. Using a more accessible sample type than sputum may enable the detection of more cases, but it is critical that alternative samples be tested appropriately. Here, we describe two new, highly accurate methods for testing tongue swabs for TB DNA.
Dengue and Zika virus NS4B proteins differ in topology and in determinants of ER membrane protein complex dependency
Authors: Porter Samuel S. , Gilchrist Talon M. , Schrodel Samantha , Tai Andrew W. 
Published: December 2024

ABSTRACT

Flaviviruses utilize the cellular endoplasmic reticulum (ER) for all aspects of their lifecycle. Genome replication and other viral activities take place in structures called replication organelles (ROs), which are invaginations induced in the ER membrane. Among the required elements for RO formation is the biogenesis of viral nonstructural proteins NS4A and NS4B. We have previously shown that NS4A and NS4B from Dengue virus (DENV) and Zika virus (ZIKV) depend on the cellular ER membrane protein complex (EMC) for biogenesis. Here, we find that this dependency extends to the NS4A and NS4B proteins of Yellow Fever virus (YFV) and West Nile virus (WNV), which share similar computationally predicted membrane topologies. However, we demonstrate that ZIKV NS4B has different determinants of its dependency on the EMC than those for DENV NS4B, as well as a different membrane topology. Furthermore, we characterize mutant isolates of DENV and ZIKV that were serially passaged in EMC knockout cells and find that none are completely independent of the EMC for infection, and that mutant NS4B proteins remain sensitive to EMC depletion, suggesting a high genetic barrier to EMC depletion. Collectively, our findings are consistent with a model in which the EMC recognizes multiple determinants in the NS4B protein to support infection in several flaviviruses of critical public health importance.

IMPORTANCE
The NS4A and NS4B proteins of flaviviruses are critically important to replication, but little is known about their function. It has been previously reported that the cellular EMC supports the biogenesis of NS4A and NS4B from Dengue and Zika virus. In this work, we demonstrate that this dependency on the EMC for NS4A and NS4B biogenesis extends to the West Nile and Yellow Fever viruses. Furthermore, we examine the features of ZIKV NS4B and find that its membrane topology of ZIKV NS4B and its determinants of dependency on the EMC are different from those previously described in DENV NS4B. Finally, we present evidence that there is a high genetic barrier for Dengue and Zika viruses to overcome EMC depletion.
Induction of PD-1 and CD44 in CD4
+
T cells by circulatory extracellular vesicles from severe dengue patients drives endothelial damage via the NF-kB signaling pathway
Authors: Kumari Sharda , Biswas Ankit , Maiti Tushar Kanti , Bandyopadhyay Bhaswati , Banerjee Arup 
Published: December 2024

ABSTRACT

Extracellular vesicles (EVs) emerged as critical contributors to the pathogenesis of vascular endothelial barrier dysfunction during the inflammatory response to infection. However, the contribution of circulating EVs to modifying endothelial function during dengue virus infection remains unclear. In this study, we showed that severe dengue patients’ plasma-derived EV (SD-EV) were found to carry elevated levels of different protein cargos, e.g., immunoregulatory proteins (PD-L1, CD44). Further, we demonstrated that SD-EV induces PD-1 and CD44 expression on CD4
+
T cells. SD-EV-modulated CD4
+
T (SD-EV-CD4) cells released secretome delayed endothelial cell (EC) migration, arrested them in the G1 phase, and augmented the expression of PD-L1 and ICAM-1 expression on EC through the Notch signaling pathway. Blocking SD-EV and CD4
+
T-cell interaction through the PD-1/PD-L1 pathway partially rescued the CD4
+
T cell’s effect on EC but did not alter ICAM-1 expression on EC. We observed that the ICAM-1 expression on EC and hyaluronic acid (HA) release from EC was mediated by CD44, which was elevated on SD-EV-modulated CD4
+
T cells (SD-EV-CD4), indicating a permeability defect. Blocking of CD44 on SD-EV-CD4 significantly reduced ICAM-1 expression on EC. Further, depletion of specific cytokines, e.g., TNF-α and not IFN-γ from the SD-EV-CD4 secretome, reduced ICAM-1 expression, decreased transendothelial electrical resistance, and induced apoptosis on EC significantly. Treatment with NF-kB inhibitor before secretome addition to EC reduced ICAM-1 expression on EC. In conclusion, we provided evidence that SD-EV-CD4 carrying PD-1 and CD44, when interacting with EC, significantly affected endothelial cell properties and may be significant in dengue-mediated endothelial dysfunction.

IMPORTANCE

Extracellular vesicles (EVs) are small membrane vesicles secreted into biological fluids, including plasma from living cells, holding insights into pathological processes. Studying EVs under pathological conditions is extremely important as they play a selective role in intercellular communication and modulation of immune response under diverse pathological conditions. However, there is less clarity on how circulatory extracellular vesicles influence immune cells during dengue virus (DV) infection and impact pathogenesis. Our present study highlights the impact of severe dengue patients’ plasma-derived EV (SD-EV) on CD4
+
T cells and together induce endothelial barrier dysfunction. We provided evidence that SD-EV induces PD-1 and CD44 on CD4
+
T cells and, when interacting with endothelial cells (EC), drives endothelial damage through direct interaction or secretome and may be significant in dengue-mediated endothelial dysfunction.
YBX1 is required for assembly of viral replication complexes of chikungunya virus and replication of multiple alphaviruses
Authors: Li Zhen-Qi , Zhao Li-Xin , Wang Su-Yun , Hu Chu-Yu , Wang Yan-Yi , Yang Yan 
Published: December 2024

ABSTRACT

Chikungunya virus (CHIKV), an enveloped positive-sense RNA virus, is a member of the alphaviruses and cause fever and arthralgia in humans. We performed genome-wide CRISPR/Cas9-based screens and identified Y-box binding protein 1 (YBX1) as an essential cellular factor for CHIKV. Deficiency of YBX1 inhibited CHIKV RNA replication and impaired virus production. Upon CHIKV infection, YBX1 showed a striking re-localization to viral replication complexes (vRCs), where it co-localized with CHIKV nsP3 and dsRNA intermediates. YBX1 directly interacted with CHIKV nsP3, and mutation of the YBX1-binding motif in CHIKV nsP3 suppressed viral replication in host cells. Furthermore, YBX1 bound to viral RNA and increased the viral RNA-binding activity of CHIKV nsP3. Consistently, the RNA-binding activity of YBX1, as well as the ability of nsP3 to bind to YBX1, was required for efficient CHIKV replication. In addition to CHIKV, YBX1 was also essential for replication of all examined alphaviruses including the prototypic alphavirus. Our findings suggest that YBX1 acts as a scaffold for assembly of chikungunya vRCs and an important factor for replication of multiple alphaviruses, which may serve as a potential target for the development of anti-alphavirus therapies.

IMPORTANCE

Alphaviruses are a group of mosquito-transmitted, enveloped, positive-strand RNA viruses in the
Togaviridae
family. Most alphaviruses are important pathogens that continue to cause human disease ranging from severe and potentially fatal neurological disease to chronic arthritic disease on a global scale. Here, we found that YBX1 promotes binding of CHIKV genomic RNA to nsP3, which is a key component of the replication complex, and is therefore pivotal for CHIKV replication. Deficiency of YBX1 results in reduced replication of multiple alphaviruses, including arthritogenic and encephalitic alphaviruses. These findings suggest that YBX1 is an important cellular factor for multiple alphaviruses and a potential target for preventing alphavirus infections.
Discovery of small molecules against porcine reproductive and respiratory syndrome virus replication by targeting NendoU activity
Authors: Zhu Jiaqi , Lai Yunqiang , Cheng Mengqi , Dash Radha Charan , Guo Shuangshuang , Guo Jintong , Su Yue , Wolek Andrew , Issacs Brianna , Liu Zhenming , Li Qi , Mishra Neha , Garmendia Antonio , Hadden M. Kyle , Tian X. Cindy , He Xin , Tang Young 
Published: December 2024

ABSTRACT

Porcine reproductive and respiratory syndrome (PRRS) remains a major threat to animal health and causes substantial economic losses worldwide. The nonstructural protein 11 (NSP11) of the causative agent, PRRS virus (PRRSV), contains a highly conserved nidoviral uridylate-specific endoribonuclease (NendoU) domain essential for viral replication and immune evasion. Targeting NSP11 offers a novel approach to antiviral intervention. Through
in silico
virtual screening followed by a fluorescence resonance energy transfer assay , we identified A8-A2 as a promising candidate that effectively inhibits NendoU activity. Molecular docking and mutational analysis revealed that A8-A2 and its analogs target the key catalytic residues His144 and Thr217 of NSP11, located within the NendoU enzyme activity loop and pocket region, respectively. A8-A2 demonstrated dose-dependent inhibition of PRRSV replication in porcine alveolar macrophages. Notably, the NendoU is conserved across PRRSV strains and other Nidoviruses, and A8-A2 exhibited antiviral activity against both type I and type II PRRSV strains, as well as the infectious bronchitis virus, a coronavirus in the order
Nidovirales
. Further investigations revealed that A8-A2 impedes viral replication early in infection and reverses NSP11-mediated suppression of Poly(I:C)-induced interferon production. However, this effect occurs independently of mRNA splicing inhibition. These findings indicate that A8-A2 could act as an effective antiviral agent against infections caused by diverse PRRSV strains and may serve as a broad-spectrum agent for other Nidoviruses.

IMPORTANCE
Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the pig industry, and vaccination is the principal method to prevent this viral infection currently. However, vaccination often fails to provide protection against heterologous strains, highlighting the need for alternative strategies for broad protection. The nidoviral uridylate-specific endoribonuclease (NendoU) domain plays a crucial role in viral replication and evasion of host immune responses. In this study, we identified a group of new compounds with similar chemical structures that could interfere with NendoU enzyme activity. Among these compounds, A8-A2 significantly inhibited PRRSV replication in host cells with minimal cytotoxicity. Our findings provide a new direction for developing potent antiviral compounds that can offer broad protection against different PRRSV strains, thereby mitigating their impact on pig health and benefiting the husbandry industry.
Draft whole-genome sequence of
Enterococcus faecium
isolate from a clinical sample from Dhaka, Bangladesh
Authors: Towhid Syeda Tasneem , Shihab Md Maksudur Rahman , Jubair Mohammad , Faisal Naimul Islam , Rahman Md. Mustafizur 
Published: December 2024

ABSTRACT

We collected a urine specimen from a 20-year-old male, cultured it on Chrom-agar
Enterococcus
, identified it with VITEK2, and whole-genome sequenced it using the PacBio RSII. The genome was 3,111,621 nucleotides long with a GC% of 37.6%. It was identified as a human pathogen with a 92.3% probability and is available on NCBI with an accession ID of
JBFBPS000000000.1
.
A novel robust network construction and analysis workflow for mining infant microbiota relationships
Authors: Jiang Wei , Zhai Yue , Chen Dongbo , Yu Qinghua 
Published: December 2024

ABSTRACT

The gut microbiota plays a crucial role in infant health, with its development during the first 1,000 days influencing health outcomes. Understanding the relationships within the microbiota is essential to linking its maturation process to these outcomes. Several network-based methods have been developed to analyze the developing patterns of infant microbiota, but evaluating the reliability and effectiveness of these approaches remains a challenge. In this study, we created a test data pool using public infant microbiome data sets to assess the performance of four different network-based methods, employing repeated sampling strategies. We found that our proposed Probability-Based Co-Detection Model (PBCDM) demonstrated the best stability and robustness, particularly in network attributes such as node counts, average links per node, and the positive-to-negative link (P/N) ratios. Using the PBCDM, we constructed microbial co-existence networks for infants at various ages, identifying core genera networks through a novel network shearing method. Analysis revealed that core genera were more similar between adjacent age ranges, with increasing competitive relationships among microbiota as the infant microbiome matured. In conclusion, the PBCDM-based networks reflect known features of infant microbiota and offer a promising approach for investigating microbial relationships. This methodology could also be applied to future studies of genomic, metabolic, and proteomic data.

IMPORTANCE
As a research method and strategy, network analysis holds great potential for mining the relationships of bacteria. However, consistency and solid workflows to construct and evaluate the process of network analysis are lacking. Here, we provide a solid workflow to evaluate the performance of different microbial networks, and a novel probability-based co-existence network construction method used to decipher infant microbiota relationships. Besides, a network shearing strategy based on percolation theory is applied to find the core genera and connections in microbial networks at different age ranges. And the PBCDM method and the network shearing workflow hold potential for mining microbiota relationships, even possibly for the future deciphering of genome, metabolite, and protein data.
RamA upregulates the ATP-binding cassette transporter
mlaFEDCB
to mediate resistance to tetracycline-class antibiotics and the stability of membranes in
Klebsiella pneumoniae
Authors: Zhao Xiaoyu , Zhang Yixin , Ju Mohan , Yang Yang , Liu Haoqi , Qin Xiaohua , Xu Qingqing , Hao Min 
Published: December 2024

ABSTRACT

RamA is an intrinsic regulator in
Klebsiella pneumoniae
, belonging to the AraC family of transcription factors and conferring a multidrug resistance phenotype, especially for tetracycline-class antibiotics. The ATP-binding cassette transporters MlaFEDCB in bacteria play essential roles in functions essential for cell survival and intrinsic resistance to many antibiotics. We found deletion of
ramA
resulted in a fivefold decrease in the transcriptional levels of the
mlaFEDCB
operon. After complementation with ramA, the transcriptional levels were comparable to those of wild-type strain. Furthermore, an electrophoretic mobility shift assay showed that RamA could bind to the promoter region of
mlaEFDCB
operon, which confirmed RamA is an activator of
mlaEFDCB
operon. The
mlaEFDCB
operon could mildly mediate resistance to the tetracycline family of antibiotics under RamA regulation. The MIC (minimum inhibitory concentration) of tigecycline decreased fourfold, and the MIC of doxycycline, minocycline, and eravacycline decreased twofold after
mlaE
-knockout. The
ramA
- and
mlaE
-knockout strains exhibited greater sensitivity to sodium dodecyl sulfate (SDS)-EDTA than the wild-type. Growth of Δ
ramA
cells was severely compromised in 0.25/0.5% SDS and 0.55 mM EDTA, and this sensitivity was restored by complementation with
ramA
and
mlaE
. This study demonstrates that RamA can directly regulate the
malEFEDCB
operon, thereby mediating resistance to tetracycline-class antibiotics, contributing to the stability of bacterial membranes in
K. pneumoniae
. We identified a novel signal pathway in which RamA mediates multidrug resistance of
K. pneumoniae
, leading to new ideas for the development of novel antimicrobial therapeutics, therefore deserving further comprehensive study.

IMPORTANCE

Multidrug-resistant and extensively drug-resistant
Klebsiella pneumoniae
have emerged as significant global health concerns resulting in high mortality rates. Although previous research has investigated the maintenance of lipid asymmetry (Mla) pathway, the extent to which it mediates antimicrobial resistance in
K. pneumoniae
and the underlying upstream regulatory mechanisms remain unclear. In this study, we sought to determine at the molecular level how the AraC-type global regulator RamA directly regulates mlaFEDCB, which mediates resistance to tetracycline-class antibiotics and the stability of bacterial membranes in
K. pneumoniae
.
Mathematical modelling and analysis for the co-infection of viral and bacterial diseases: a systematic review protocol
Authors: Yano Timothy Kiprono , Afrifa-Yamoah Ebenezer , Collins Julia , Mueller Ute , Richardson Steven 
Published: December 2024

IntroductionBreaking the chain of transmission of an infectious disease pathogen is a major public health priority. The challenges of understanding, describing and predicting the transmission dynamics of infections have led to a wide range of mathematical, statistical and biological research problems. Advances in diagnostic laboratory procedures with the ability to test multiple pathogens simultaneously mean that co-infections are increasingly being detected, yet little is known about the impact of co-infections in shaping the course of an infection, infectivity, and pathogen replication rate. This is particularly true of the apparent synergistic effects of viral and bacterial co-infections, which present the greatest threats to public health because of their lethal nature and complex dynamics. This systematic review protocol is the foundation of a critical review of co-infection modelling and an assessment of the key features of the models.Methods and analysisMEDLINE through PubMed, Web of Science, medRxiv and Scopus will be systematically searched between 1 December 2024 and 31 January 2025 for studies published between January 1980 and December 2024. Three reviewers will screen articles independently for eligibility, and quality assessment will be performed using the TRACE (TRAnsparent and Comprehensive Ecological) standard modelling guide. Data will be extracted using an Excel template in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis standard reporting guidelines. This systematic review will apply the SWiM (Synthesis Without Meta-analysis) approach in its narrative synthesis coupled with tables and figures to present data. The synthesis will highlight key dynamical co-infection model features such as assumptions, data fitting and estimation methods, validation and sensitivity analyses, optimal control analyses, and the impact of co-infections.Ethics and disseminationEthics approval is not required for a systematic review since it will be based on published work. The output of this study will be submitted for publication in a peer-reviewed journal.PROSPERO registration numberCRD42023481247.