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Enhanced editing of Bifidobacterium lactis using the endogenous Type I-G CRISPR-Cas system.

Diverse Bifidobacterium animalis subsp. lactis strains are widely used as commercial probiotics. While proof-of-concept studies have shown t … idate a broadly applicable genome editing framework. We redesigned backbone plasmids with different replicons and antibiotic resistance markers and evaluated performance across six commercial strains for transformation efficiency. A vector carrying the pBC1 origin coupled with a chloramphenicol resistance marker improved transformation in most strains. Usi …

Comparative performance of portable DNA extraction protocols and bioinformatics workflows for rapid detection of gram-negative bacteria and antimicrobial resistance using Oxford Nanopore sequencing.

Oxford Nanopore Technology (ONT) enables rapid, portable pathogen identification and antimicrobial resistance (AMR) detection, but the relia … and enabled a broader, more complete detection of virulence factors, pathogenicity islands, plasmid replicons, and accessory AMR genes, reflecting enhanced genomic resolution.IMPORTANCERapid whole-genome sequencing is increasingly used to detect antimicrobial resistance and guide public health responses, but its reliability depends strongly on how bacteria …

Whole-genome sequencing analysis of clinical carbapenemase-producing bacteria isolated in Dakar, Senegal.

Antimicrobial resistance, particularly to carbapenems, represents a major global public health threat. In Senegal, the increasing prevalence … disk diffusion testing. Whole-genome sequencing enabled the detection of resistance genes, plasmid replicons, and clonal lineages through MLST analysis. Among the 50 isolates, 35 (70%) carried at least one carbapenemase gene. The blaNDM-5 gene was the most frequent (15/35; 42.9%), followed by blaOXA-181 (20.0%), blaNDM-1 (14.3%), and blaOXA-484 (11.4%). C …

Multidrug resistance and genomic characteristics of nontypeable Haemophilus influenzae isolates from the respiratory tract of pediatric patients.

UNLABELLED: Nontypeable Haemophilus influenzae (NTHi) is a common colonizer of the human upper respiratory tract and one of the major pathog … laTEM-1 carriage (77.9%), with minor contributions from chromosomal ftsI mutations. Fifteen plasmid replicons were predicted from 25 isolates, which highly coincided with the carriage of blaTEM-1 and other acquired resistance genes. This study demonstrates that MDR in pediatric NTHi is primarily driven by acquired resistance genes and chromosomal mutations …

Development of a GFP-labeled Pasteurella multocida strain for in vivo organ infection tracing using a stable RCR-based genetic manipulation plasmid.

UNLABELLED: Advances in microscopy have enabled the widespread application of in vivo pathogen tracking approaches to bacterial pathogenesis … evaluated diverse replication mechanisms and identified that rolling circle replication (RCR)-type replicons exhibit superior transformation robustness in swine isolates. Leveraging the temperature-sensitive pSET4s backbone and an optimized mpheS counter-selection marker, we developed a high-efficiency, markerless genetic manipulation system. The system e …

Patterns of antimicrobial resistance genes in pathogens across One Health sectors in Ireland: an in silico approach.

As part of a rapid risk assessment, an in silico approach was used to detect antimicrobial resistance (AMR) in pathogenic isolates from huma … dentified 799 antimicrobial resistance genes (ARGs), including their allelic diversity, 117 plasmid replicons, and 274 virulence factors. Several critical ARGs, particularly those conferring resistance to beta-lactams, aminoglycosides, quinolones, and colistin, were common across isolates originating from human, animal, and environmental sources, suggestin …

No evidence of multidrug-resistant Enterobacterales transmission between healthy companion animals and pet owners in the greater Atlanta area: a pilot study.

Antimicrobial resistance (AMR) is a global concern affecting both animals and humans. Pets share a close bond with humans and are exposed to … fferent resistance genes in E. coli isolates from pets and eight from humans, six different plasmid replicons, and all were located in four different phylogroups. Phylogenetic analysis indicates species-specific clustering based on host. Our results demonstrate that while MDR Enterobacterales were present in both humans and their pets in this Atlanta popul …

Characterization of Enterobacter cloacae complex clinical isolates: comparative genomics and the role of the efflux pump AcrAB-TolC over-expression and NDM-1 production.

BACKGROUND: Carbapenem-resistant Enterobacter cloacae complex (ECC) has emerged as the third most prevalent nosocomial Enterobacterales path … at 3-8×10-3 per donor and were embedded in Tn125/Tn3000 transposons flanked by ISAba125/IS26. These replicons also carried ESBLs (bla CTX-M-15), bla TEM-1, aminoglycoside- modifying enzymes (armA, aadA1) and intact class 1 integrons (In2, In799 and In1465). Despite>90% backbone identity of the bla NDM-1-ble MBL, the module could be reversibly excised, inve …

Genomic characterization of an ESBL-producing Klebsiella pneumoniae ST37 recovered from a hospitalized patient in Armenia.

Multidrug-resistant Klebsiella pneumoniae continues to challenge healthcare services globally. However, our understanding of its occurrence, … , irp2, ybtAEPQSTUX, and fyuA). Moreover, we also detected five insertion sequences and two plasmid replicons in the ARM02 genome. Phylogenetic analysis showed that ARM02 shared a common ancestor with the USA strains SRR5283489 and SRR5973349, diverging around 2007 (95% onfidence interval, 2004 to 2011). However, ARM02 carried a unique accessory genome, in …

Comparative genomic insights into multidrug resistance in classical and hypervirulent K. pneumoniae clinical isolates.

Klebsiella pneumoniae is a highly adaptable opportunistic pathogen responsible for various infections, particularly in immunocompromised ind … actamases (DHA-1, TEM-1D, CMH-1, CMY-6, LAP-2, AmpC1) were also present. The most prevalent plasmid replicons were Col(pHAD28), IncFIB(K), and IncR, with the hypervirulent ST147-1LV isolate carrying the highest number. These findings underscore the significant public health threat posed by hypervirulent and MDR K. pneumoniae strains, highlighting the exten …

Escherichia coli ST117: exploring the zoonotic hypothesis.

UNLABELLED: Extraintestinal pathogenic Escherichia coli (ExPEC) can lead to severe infections, with additional risks of increasing antimicro … 61) frequently carrying resistance to critical antibiotics (>86%) linked to IncFII, IncI1, or IncX4 replicons. High similarities were found between IncX4 from Brazilian outbreaks and those from E. coli recovered from imported Brazilian poultry meat and human clinical cases. The ST117 phylogeny showed non-specificity according to host and continent and an A …

Genomic Characterization of ESBL/AmpC-Producing Escherichia coli in Stray Dogs Sheltered in Yangzhou, China.

PURPOSE: Limited data are available on the prevalence and antimicrobial resistance of extended spectrum β-lactamase- (ESBL) and AmpC β-lacta … lowed by phylogroup B1 (26.3%, n=21). Thirty different sequence types (STs) and 27 distinct plasmid replicons were identified, among which ST2325 (n=12) and IncFII (n=38) was the most frequent ST and plasmid, respectively. ESBL/AmpC-producing isolates were divided into four major clades; clade IV was the primary lineage containing 37 isolates from feces an …

Prevalence of Cefotaxime-Resistant Escherichia coli Isolates from Healthy Cattle and Sheep in Northern Spain: Phenotypic and Genome-Based Characterization of Antimicrobial Susceptibility.

In order to estimate herd-level prevalence of extended-spectrum β-lactamase/AmpC β-lactamase (ESBL/AmpC)- and carbapenemase-producing commen … er. The plasmid/chromosomal locations of resistance genes were predicted with PlasFlow, and plasmid replicons were identified using PlasmidFinder. Fifty-two acquired resistance genes and point mutations in another four genes that coded for resistance to 11 antimicrobial classes were identified. Fifty-five genomes carried ESBL-encoding genes, blaCTX-M-14 be …

Functional Consequences of RNA 5'-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation.

Group B coxsackieviruses are responsible for chronic cardiac infections. However, the molecular mechanisms by which the virus can persist in … xsackievirus B3 biology. Using cell extracts from HeLa cells, as well as transfection of luciferase replicons in two types of cardiomyocytes, we demonstrated that coxsackievirus RNAs harboring 5' deletions ranging from 7 to 49 nucleotides in length can be translated nearly as efficiently as those of wild-type virus. However, these 5' deletions greatly redu …

Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase.

Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised in … oma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-H …

Virus-Like Vesicle-Based Therapeutic Vaccine Vectors for Chronic Hepatitis B Virus Infection.

UNLABELLED: More than 500,000 people die each year from the liver diseases that result from chronic hepatitis B virus (HBV) infection. Therapeutic vaccines, which aim to elicit an immune response capable of controlling the virus, offer a potential new treatment strategy for chronic hepatitis B. Recently, an evolved, high-titer vaccine platform consisting of Semliki Forest virus RNA replicons that express the vesicular stomatitis virus glycoprotein (VSV G) has been described. This platform generates virus-like vesicles (VLVs) that contain VSV G but no other viral structural proteins. We report …

Clonal spread of CTX-M-15-producing Klebsiella pneumoniae in a Croatian hospital.

This study was conducted to detect and analyse the presence of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae assoc … 5) gene, which has recently been demonstrated to mobilize 3'-adjacent genes to transfer between DNA replicons. The isolates contained a large plasmid of approximately 150 kb. The isolates were assigned to five clusters (>85 % similarity), which contained subclusters. The results of this work provided insights into the molecular epidemiology of the spread o …

Viral Vector-Based Melanoma Gene Therapy.

Gene therapy applications of oncolytic viruses represent an attractive alternative for cancer treatment. A broad range of oncolytic viruses, including adenoviruses, adeno-associated viruses, alphaviruses, herpes simplex viruses, retroviruses, lentiviruses, rhabdoviruses, reoviruses, measles virus, Newcastle disease virus, picornaviruses and poxviruses, have been used in diverse preclinical and clinical studies for the treatment of various diseases, including colon, head-and-neck, prostate and breast cancer as well as squamous cell carcinoma and glioma. The majority of studies have focused on i

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… Alphavirus DNA replicon vectors have been evaluated in animal models. … In another approach, a SIN RNA replicon expressing the rabies virus glycoprotein gene was applied for immunization of mice using only 10 µg of SIN-Rab-G RNA [ 183 ]. …

The Old World and New World alphaviruses use different virus-specific proteins for induction of transcriptional shutoff.

Alphaviruses are widely distributed throughout the world. During the last few thousand years, the New World viruses, including Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), evolved separately from those of the Old World, i.e., Sindbis virus (SINV) and Semliki Forest virus (SFV). Nevertheless, the results of our study indicate that both groups have developed the same characteristic: their replication efficiently interferes with cellular transcription and the cell response to virus replication. Transcriptional shutoff caused by at least two of the Old

Translation and replication of hepatitis C virus genomic RNA depends on ancient cellular proteins that control mRNA fates.

Inevitably, viruses depend on host factors for their multiplication. Here, we show that hepatitis C virus (HCV) RNA translation and replication depends on Rck/p54, LSm1, and PatL1, which regulate the fate of cellular mRNAs from translation to degradation in the 5'-3'-deadenylation-dependent mRNA decay pathway. The requirement of these proteins for efficient HCV RNA translation was linked to the 5' and 3' untranslated regions (UTRs) of the viral genome. Furthermore, LSm1-7 complexes specifically interacted with essential cis-acting HCV RNA elements located in the UTRs. These results bridge HCV