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506800.9336Ultrasensitive Label-Free Detection of Unamplified Multidrug-Resistance Bacteria Genes with a Bimodal Waveguide Interferometric Biosensor. Infections by multidrug-resistant bacteria are becoming a major healthcare emergence with millions of reported cases every year and an increasing incidence of deaths. An advanced diagnostic platform able to directly detect and identify antimicrobial resistance in a faster way than conventional techniques could help in the adoption of early and accurate therapeutic interventions, limiting the actual negative impact on patient outcomes. With this objective, we have developed a new biosensor methodology using an ultrasensitive nanophotonic bimodal waveguide interferometer (BiMW), which allows a rapid and direct detection, without amplification, of two prevalent and clinically relevant Gram-negative antimicrobial resistance encoding sequences: the extended-spectrum betalactamase-encoding gene blaCTX-M-15 and the carbapenemase-encoding gene blaNDM-5 We demonstrate the extreme sensitivity and specificity of our biosensor methodology for the detection of both gene sequences. Our results show that the BiMW biosensor can be employed as an ultrasensitive (attomolar level) and specific diagnostic tool for rapidly (less than 30 min) identifying drug resistance. The BiMW nanobiosensor holds great promise as a powerful tool for the control and management of healthcare-associated infections by multidrug-resistant bacteria.202033086716
786810.9333A double-quenching paperclip ECL biosensing platform for ultrasensitive detection of antibiotic resistance genes (mecA) based on Ti(3)C(2) MXene-Au NPs as a coreactant accelerator. The global spread of environmental biological pollutants, such as antibiotic-resistant bacteria and their antibiotic resistance genes (ARGs), has emerged as a critical public health concern. It is imperative to address this pressing issue due to its potential implications for public health. Herein, a DNA paperclip probe with double-quenching function of target cyclic cleavage was proposed, and an electrochemiluminescence (ECL) biosensing platform was constructed using Ti(3)C(2) MXene in-situ reduction growth of Au NPs (TCM-Au) as a coreactant accelerator, and applied to the sensitive detection of ARGs. Thanks to the excellent catalytic performance, large surface area and Au-S affinity of TCM-Au, the ECL performance of CdS QDs have been significantly improved. By cleverly utilizing the negative charge of the paperclip nucleic acid probe and its modification group, double-quenching of the ECL signal was achieved. This innovative approach, combined with target cyclic amplification, facilitated specific and sensitive detection of the mecA gene. This biosensing platform manifested highly selective and sensitive determination of mecA genes in the range of 10 fM to 100 nM and a low detection limit of 2.7 fM. The credible detectability and anti-interference were demonstrated in Yangtze river and Aeration tank outlet, indicating its promising application toward pollution monitoring of ARGs.202337666010
999520.9276Direct fluorescence in situ hybridization (FISH) in Escherichia coli with a target-specific quantum dot-based molecular beacon. Quantum dots (QDs) are inorganic fluorescent nanocrystals with excellent properties such as tunable emission spectra and photo-bleaching resistance compared with organic dyes, which make them appropriate for applications in molecular beacons. In this work, quantum dot-based molecular beacons (QD-based MBs) were fabricated to specifically detect β-lactamase genes located in pUC18 which were responsible for antibiotic resistance in bacteria Escherichia coli (E. coli) DH5α. QD-based MBs were constructed by conjugating mercaptoacetic acid-quantum dots (MAA-QDs) with black hole quencher 2 (BHQ2) labeled thiol DNA vial metal-thiol bonds. Two types of molecular beacons, double-strands beacons and hairpin beacons, were observed in product characterization by gel electrophoresis. Using QD-based MBs, one-step FISH in tiny bacteria DH5α was realized for the first time. QD-based MBs retained their bioactivity when hybridizing with complementary target DNA, which showed excellent advantages of eliminating background noise caused by adsorption of non-specific bioprobes and achieving clearer focus of genes in plasmids pUC18, and capability of bacterial cell penetration and signal specificity in one-step in situ hybridization.201020729070
10130.9269The encapsulated strain TIGR4 of Streptococcus pneumoniae is phagocytosed but is resistant to intracellular killing by mouse microglia. The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD(50) of 2 × 10² CFU, whereas the LD(50) for the unencapsulated FP23 was greater than 10⁷ CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing.201020615478
583540.9260Rapid and Ultrasensitive Detection of Mutations and Genes Relevant to Antimicrobial Resistance in Bacteria. The worldwide emergence of multidrug-resistant (MDR) bacteria is associated with significant morbidity, mortality, and healthcare costs. Rapid and accurate diagnostic methods to detect antibiotic resistance are critical for antibiotic stewardship and infection control measurements. Here a cantilever nanosensor-based diagnostic assay is shown to detect single nucleotide polymorphisms (SNPs) and genes associated with antibiotic resistance in Gram negative (Pseudomonas aeruginosa) and positive (Enterococcus faecium) bacteria, representing frequent causes for MDR infections. Highly specific RNA capture probes for SNPs (ampR(D135G) or ampR(G154R) ) or resistance genes (vanA, vanB, and vanD) allow to detect the binding of bacterial RNA within less than 5 min. Serial dilutions of bacterial RNA indicate an unprecedented sensitivity of 10 fg µL(-1) total RNA corresponding to less than ten bacterial cells for SNPs and 1 fg µL(-1) total RNA for vanD detection equivalent to single bacterial cell sensitivity.202133552553
815550.9260Gut bacteria enable prostate cancer growth. Testosterone-synthetizing gut bacteria drive resistance to therapy.202134618567
843260.9259A 0D-2D Heterojunction Bismuth Molybdate-Anchored Multifunctional Hydrogel for Highly Efficient Eradication of Drug-Resistant Bacteria. Due to the increasing antibiotic resistance and the lack of broad-spectrum antibiotics, there is an urgent requirement to develop fresh strategies to combat multidrug-resistant pathogens. Herein, defect-rich bismuth molybdate heterojunctions [zero-dimensional (0D) Bi(4)MoO(9)/two-dimensional (2D) Bi(2)MoO(6), MBO] were designed for rapid capture of bacteria and synergistic photocatalytic sterilization. The as-prepared MBO was experimentally and theoretically demonstrated to possess defects, heterojunctions, and irradiation triple-enhanced photocatalytic activity for efficient generation of reactive oxygen species (ROS) due to the exposure of more active sites and separation of effective electron-hole pairs. Meanwhile, dopamine-modified MBO (pMBO) achieved a positively charged and rough surface, which conferred strong bacterial adhesion and physical penetration to the nanosheets, effectively trapping bacteria within the damage range and enhancing ROS damage. Based on this potent antibacterial ability of pMBO, a multifunctional hydrogel consisting of poly(vinyl alcohol) cross-linked tannic acid-coated cellulose nanocrystals (CPTB) and pMBO, namely CPTB@pMBO, is developed and convincingly effective against methicillin-resistant Staphylococcus aureus in a mouse skin infection model. In addition, the strategy of combining a failed beta-lactam antibiotic with CPTB@pMBO to photoinactivation with no resistance observed was developed, which presented an idea to address the issue of antibiotic resistance in bacteria and to explore facile anti-infection methods. In addition, CPTB@pMBO can reduce excessive proteolysis of tissue and inflammatory response by regulating the expression of genes and pro-inflammatory factors in vivo, holding great potential for the effective treatment of wound infections caused by drug-resistant bacteria.202337531599
843770.9257Tocopherol polyethylene glycol succinate-modified hollow silver nanoparticles for combating bacteria-resistance. Multiple drug resistance and the increase in the appearance of superbugs together with the exceedingly scant development of new potent antibiotic drugs pose an urgent global medical threat and imminent public security crisis. In the present study, we fabricated well-dispersed tocopherol polyethylene glycol succinate (TPGS)-capped silver nanoparticles (AgNPs) of about 10 nm in size. The hollow structure of the TPGS-capped AgNPs (TPGS/AgNPs) was confirmed and applied to load antibiotics. The TPGS/AgNPs proved to be able to cross the bacterial cell wall and penetrate into bacteria, thereby delivering more of the antibiotic to the interior of bacteria and thus enhancing the in vitro antibacterial effect of the antibiotic, even overcoming the drug-resistance in drug-resistant E. coli and Acinetobacter baumannii. It was found that the TPGS modification in the TPGS/AgNPs could decrease the activity of the efflux pumps AdeABC and AdeIJK in drug-resistant Acinetobacter baumannii via inhibiting the efflux pump genes adeB and adeJ, thus increasing the accumulation of the delivered antibiotic and overcoming the drug-resistance. Tigecycline delivered by TPGS/AgNPs could effectively antagonize drug-resistance in an acute peritonitis model mice, thereby increasing the survival rate and alleviating the inflammatory response. TPGS/AgNPs were developed as a novel and effective antibiotic delivery system and TPGS was demonstrated to have great potential as a pharmaceutical excipient for use in drug-resistant infection therapy.201930968093
247480.9255Nano-biosensors for rapid detection of antibiotic resistance genes blaCTX-M in Escherichia coli and blaKPC in Klebsiella pneumoniae. The wide spread of antibiotic resistance genes in clinical isolates of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) has become a worrying issue worldwide. Thus, electrochemical DNA-based biosensors were developed and applied for the rapid detection of antibiotic resistance genes (blaCTX-M extended-spectrum β-lactamase (ESBL) in E. coli and blaKPC carbapenemase in Klebsiella pneumoniae). Selective ssDNA probes specific to the targeting complementary genes were designed and individually immobilized onto disposable sensor chips that were functionalized with nanocomposite (ssDNA-probe@ATP@AuNPs-PPY-V(2)O(5)). In this regard, the sensor surfaces were first modified with vanadium oxide (V(2)O(5)) and polypyrrole (PPy) then gold nanoparticles (AuNPs) to effectively promote the chemical immobilization of each of the designed bio-receptor (ssDNA probe) onto the sensor matrix using the 4-aminothiophenol (ATP) as the chemical cross-linker. Different parameters that affect the biosensing performance were studied and optimized. Accordingly, high sensitivity to both E. coli blaCTX-M and K. pneumoniae blaKPC genes were obtained with a dynamic linear range from 10(-6) to 0.1 ng/μL with correlation coefficient (R(2)) of 0.991 and 0.988, limit of detection (LODs) of 0.5 × 10(-7) ng/μL and 1 × 10(-7) ng/μL, and limit of quantification (LOQs) of 1.2 × 10(-7) and 3 × 10(-7) ng/μL, respectively. High selectivity was provided since no electrochemical signals were obtained from various competitors of common bacteria. Finally, different types of clinical samples were analyzed and validated.202540250654
974690.9253Fluoroamphiphilic polymers exterminate multidrug-resistant Gram-negative ESKAPE pathogens while attenuating drug resistance. ESKAPE pathogens are a panel of most recalcitrant bacteria that could "escape" the treatment of antibiotics and exhibit high incidence of drug resistance. The emergence of multidrug-resistant (MDR) ESKAPE pathogens (particularly Gram-negative bacteria) accounts for high risk of mortality and increased resource utilization in health care. Worse still, there has been no new class of antibiotics approved for exterminating the Gram-negative bacteria for more than 50 years. Therefore, it is urgent to develop novel antibacterial agents with low resistance and potent killing efficacy against Gram-negative ESKAPE pathogens. Herein, we present a class of fluoropolymers by mimicking the amphiphilicity of cationic antimicrobial peptides. Our optimal fluoroamphiphilic polymer (PD(45)HF(5)) displayed selective antimicrobial ability for all MDR Gram-negative ESAKPE pathogens, low resistance, high in vitro cell selectivity, and in vivo curative efficacy. These findings implied great potential of fluoroamphiphilic cationic polymers as promising antibacterial agents against MDR Gram-negative ESKAPE bacteria and alleviating antibiotic resistance.202439196947
9981100.9253High-contrast imaging of cellular non-repetitive drug-resistant genes via in situ dead Cas12a-labeled PCR. In situ imaging of genes of pathogenic bacteria can profile cellular heterogeneity, such as the emergence of drug resistance. Fluorescence in situ hybridization (FISH) serves as a classic approach to image mRNAs inside cells, but it remains challenging to elucidate genomic DNAs and relies on multiple fluorescently labeled probes. Herein, we present a dead Cas12a (dCas12a)-labeled polymerase chain reaction (CasPCR) assay for high-contrast imaging of cellular drug-resistant genes. We employed a syncretic dCas12a-green fluorescent protein (dCas12a-GFP) to tag the amplicons, thereby enabling high-contrast imaging and avoiding multiple fluorescently labeled probes. The CasPCR assay can quantify quinolone-resistant Salmonella enterica in mixed populations and identify them isolated from poultry farms.202439229640
1474110.9250Simple, rapid, and cost-effective modified Carba NP test for carbapenemase detection among Gram-negative bacteria. PURPOSE: Detection of carbapenemases among Gram-negative bacteria (GNB) is important for both clinicians and infection control practitioners. The Clinical and Laboratory Standards Institute recommends Carba NP (CNP) as confirmatory test for carbapenemase production. The reagents required for CNP test are costly and hence the test cannot be performed on a routine basis. The present study evaluates modifications of CNP test for rapid detection of carbapenemases among GNB. MATERIALS AND METHODS: The GNB were screened for carbapenemase production using CNP, CarbAcineto NP (CANP), and modified CNP (mCNP) test. A multiplex polymerase chain reaction (PCR) was performed on all the carbapenem-resistant bacteria for carbapenemase genes. The results of three phenotypic tests were compared with PCR. RESULTS: A total of 765 gram negative bacteria were screened for carbapenem resistance. Carbapenem resistance was found in 144 GNB. The metallo-β-lactamases were most common carbapenemases followed by OXA-48-like enzymes. The CANP test was most sensitive (80.6%) for carbapenemases detection. The mCNP test was 62.1% sensitive for detection of carbapenemases. The mCNP, CNP, and CANP tests were equally sensitive (95%) for detection of NDM enzymes among Enterobacteriaceae. The mCNP test had poor sensitivity for detection of OXA-48-like enzymes. CONCLUSION: The mCNP test was rapid, cost-effective, and easily adoptable on routine basis. The early detection of carbapenemases using mCNP test will help in preventing the spread of multidrug-resistant organisms in the hospital settings.201728966495
389120.9250Implantation of unmarked regulatory and metabolic modules in Gram-negative bacteria with specialised mini-transposon delivery vectors. Engineering of robust and safe microbial cell factories requires genetic tools somewhat different from those traditionally used for laboratory-adapted microorganisms. We took advantage of the properties of broad-host-range mini-Tn5 vectors and two regulated expression systems (LacI(Q)/P(trc) and XylS/Pm), together with FRT-flanked, excisable antibiotic resistance determinants, to generate a set of vectors for the delivery of gene(s) into the chromosome of Gram-negative bacteria. This arrangement of modular elements allows the cloning and subsequent markerless insertion of expression cargoes and leaves behind an antibiotic-sensitive host upon the action of the yeast Flp recombinase. We engineered a Pseudomonas putida KT2440 Pm::gfp strain that displayed strong fluorescence upon exposure to 3-methylbenzoate, a XylS effector, and allowed us to examine the performance of the Pm promoter at the single cell level. We also reconstructed a device for sugar transport and phosphorylation in Escherichia coli independent of the native phosphoenolpyruvate-dependent phosphotransferase system by the stable implantation of genes derived from the obligate anaerobe Zymomonas mobilis. In both cases, the information carried by the implanted genes was stably inherited in the absence of any selective pressure. Deliverable expression systems such as those described here will enhance the applicability of various Gram-negative bacteria in biocatalysis and environmental bioremediation.201322609234
5070130.9249Sequence-specific DNA solid-phase extraction in an on-chip monolith: Towards detection of antibiotic resistance genes. Antibiotic resistance of bacteria is a growing problem and presents a challenge for prompt treatment in patients with sepsis. Currently used methods rely on culturing or amplification; however, these steps are either time consuming or suffer from interference issues. A microfluidic device was made from black polypropylene, with a monolithic column modified with a capture oligonucleotide for sequence selective solid-phase extraction of a complementary target from a lysate sample. Porous properties of the monolith allow flow and hybridization of a target complementary to the probe immobilized on the column surface. Good flow-through properties enable extraction of a 100μL sample and elution of target DNA in 12min total time. Using a fluorescently labeled target oligonucleotide related to Verona Integron-Mediated Metallo-β-lactamase it was possible to extract and detect a 1pM sample with 83% recovery. Temperature-mediated elution by heating above the duplex melting point provides a clean extract without any agents that interfere with base pairing, allowing various labeling methods or further downstream processing of the eluent. Further integration of this extraction module with a system for isolation and lysis of bacteria from blood, as well as combining with single-molecule detection should allow rapid determination of antibiotic resistance.201728734608
2498140.9248Emerging carbapenemases: a global perspective. The celestial rise in antibiotic resistance among Gram-negative bacteria has challenged both the scientific and pharmaceutical sectors. The hallmark of this general increase is the unbridled dissemination of carbapenem resistance genes, namely KPC, OXA and metallo-β-lactamase variants. In particular, the media attention given to the NDM-1 metallo-β-lactamase has highlighted the global consequences of human behaviour on spreading antibiotic resistance.201021129630
8184150.9246Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria. The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.202032523110
9092160.9246Antimicrobial and Antiviral Nanofibers Halt Co-Infection Spread via Nuclease-Mimicry and Photocatalysis. The escalating spread of drug-resistant bacteria and viruses is a grave concern for global health. Nucleic acids dominate the drug-resistance and transmission of pathogenic microbes. Here, imidazolium-type poly(ionic liquid)/porphyrin (PIL-P) based electrospun nanofibrous membrane and its cerium (IV) ion complex (PIL-P-Ce) are developed. The obtained PIL-P-Ce membrane exhibits high and stable efficiency in eradicating various microorganisms (bacteria, fungi, and viruses) and decomposing microbial antibiotic resistance genes and viral nucleic acids under light. The nuclease-mimetic and photocatalytic mechanisms of the PIL-P-Ce are elucidated. Co-infection wound models in mice with methicillin-resistant S. aureus and hepatitis B virus demonstrate that PIL-P-Ce integrate the triple effects of cationic polymer, photocatalysis, and nuclease-mimetic activities. As revealed by proteomic analysis, PIL-P-Ce shows minimal phototoxicity to normal tissues. Hence, PIL-P-Ce has potential as a "green" wound dressing to curb the spread of drug-resistant bacteria and viruses in clinical settings.202438647392
2496170.9243Treatment of Bloodstream Infections Due to Gram-Negative Bacteria with Difficult-to-Treat Resistance. The rising incidence of bloodstream infections (BSI) due to Gram-negative bacteria (GNB) with difficult-to-treat resistance (DTR) has been recognized as a global emergency. The aim of this review is to provide a comprehensive assessment of the mechanisms of antibiotic resistance, epidemiology and treatment options for BSI caused by GNB with DTR, namely extended-spectrum Beta-lactamase-producing Enterobacteriales; carbapenem-resistant Enterobacteriales; DTR Pseudomonas aeruginosa; and DTR Acinetobacter baumannii.202032971809
533180.9241Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol. Four new cloning vectors have been constructed from the broad-host-range cloning vector pRK290. These vectors, pLA2901, pLA2905, pLA2910, and pLA2917, confer resistance to kanamycin and tetracycline. The latter two are cosmid derivatives of pLA2901. The new vectors can be mobilized into, and are stably maintained in, a variety of gram-negative bacteria. A Sau3A genomic bank of Methylobacterium organophilum strain xx DNA has been constructed in pLA2917, and complementation analysis, with a variety of mutants unable to grow on methanol, revealed at least five separate regions necessary for growth on methanol. Complementation analysis and Tn5 mutagenesis data suggest that at least three genes are responsible for expression of active methanol dehydrogenase.19852982796
392190.9240Stable Tagging of Rhizobium meliloti with the Firefly Luciferase Gene for Environmental Monitoring. A system for stable tagging of gram-negative bacteria with the firefly luciferase gene, luc, is described. A previously constructed fusion constitutively expressing luc from the lambdap(R) promoter was used. Stable integration into the bacterial genome was achieved by use of mini-Tn5 delivery vectors. The procedure developed was applied for tagging of representative gram-negative bacteria, such as Escherichia coli, Rhizobium meliloti, Pseudomonas putida, and Agrobacterium tumefaciens. The system permitted the detection of tagged R. meliloti in the presence of more than 10 CFU per plate without the use of any selective markers (such as antibiotic resistance genes). No significant differences in growth rates or soil survival were found between the marked strain and the wild-type strain. Studies of bioluminescent R. meliloti also revealed a good correlation between cell biomass and bioluminescence. The firefly luciferase tagging system is an easy, safe, and sensitive method for the detection and enumeration of bacteria in the environment.199316349015