Role of Efflux Pumps on Antimicrobial Resistance in Pseudomonas aeruginosa.

Antimicrobial resistance is an old and silent pandemic. Resistant organisms emerge in parallel with new antibiotics, leading to a major global public health crisis over time. Antibiotic resistance may be due to different mechanisms and against different classes of drugs. These mechanisms are usually found in the same organism, giving rise to multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacteria. One resistance mechanism that is closely associated with the emergence of MDR and XDR bacteria is the efflux of drugs since the same pump can transport different classes of drugs. In Gram-negative bacteria, efflux pumps are present in two configurations: a transmembrane protein anchored in the inner membrane and a complex formed by three proteins. The tripartite complex has a transmembrane protein present in the inner membrane, a periplasmic protein, and a porin associated with the outer membrane. In Pseudomonas aeruginosa, one of the main pathogens associated with respiratory tract infections, four main sets of efflux pumps have been associated with antibiotic resistance: MexAB-OprM, MexXY, MexCD-OprJ, and MexEF-OprN. In this review, the function, structure, and regulation of these efflux pumps in P. aeruginosa and their actions as resistance mechanisms are discussed. Finally, a brief discussion on the potential of efflux pumps in P. aeruginosa as a target for new drugs is presented.

Nanoceutical Fabric Prevents COVID-19 Spread through Expelled Respiratory Droplets: A Combined Computational, Spectroscopic, and Antimicrobial Study.

Centers for Disease Control and Prevention (CDC) warns the use of one-way valves or vents in face masks for potential threat of spreading COVID-19 through expelled respiratory droplets. Here, we have developed a nanoceutical cotton fabric duly sensitized with non-toxic zinc oxide nanomaterial for potential use as a membrane filter in the one-way valve for the ease of breathing without the threat of COVID-19 spreading. A detailed computational study revealed that zinc oxide nanoflowers (ZnO NFs) with almost two-dimensional petals trap SARS-CoV-2 spike proteins, responsible to attach to ACE-2 receptors in human lung epithelial cells. The study also confirmed significant denaturation of the spike proteins on the ZnO surface, revealing removal of the virus upon efficient trapping. Following the computational study, we have synthesized ZnO NF on a cotton matrix using a hydrothermal-assisted strategy. Electron-microscopic, steady-state, and picosecond-resolved spectroscopic studies confirm attachment of ZnO NF to the cotton (i.e., cellulose) matrix at the atomic level to develop the nanoceutical fabric. A detailed antimicrobial assay using Pseudomonas aeruginosa bacteria (model SARS-CoV-2 mimic) reveals excellent antimicrobial efficiency of the developed nanoceutical fabric. To our understanding, the nanoceutical fabric used in the one-way valve of a face mask would be the choice to assure breathing comfort along with source control of COVID-19 infection. The developed nanosensitized cloth can also be used as an antibacterial/anti CoV-2 washable dress material in general.

Development of Triboelectroceutical Fabrics for Potential Applications in Self-Sanitizing Personal Protective Equipment.

Attachment of microbial bodies including the corona virus on the surface of personal protective equipment (PPE) is found to be potential threat of spreading infection. Here, we report the development of a triboelectroceutical fabric (TECF) consisting of commonly available materials, namely, nylon and silicone rubber (SR), for the fabrication of protective gloves on the nitrile platform as model wearable PPE. A small triboelectric device (2 cm × 2 cm) consisting of SR and nylon on nitrile can generate more than 20 V transient or 41 µW output power, which is capable of charging a capacitor up to 65 V in only ∼50 s. The importance of the present work relies on the TECF-led antimicrobial activity through the generation of an electric current in saline water. The fabrication of TECF-based functional prototype gloves can generate hypochlorite ions through the formation of electrolyzed water upon rubbing them with saline water. Further, computational modelling has been employed to reveal the optimum structure and mechanistic pathway of antimicrobial hypochlorite generation. Detailed antimicrobial assays have been performed to establish effectiveness of such TECF-based gloves to reduce the risk from life-threatening pathogen spreading. The present work provides the rationale to consider the studied TECF, or other materials with comparable properties, as a material of choice for the development of self-sanitizing PPE in the fight against microbial infections including COVID-19.

Effectors of the Stenotrophomonas maltophilia Type IV Secretion System Mediate Killing of Clinical Isolates of Pseudomonas aeruginosa.

Previously, we documented that Stenotrophomonas maltophilia encodes a type IV secretion system (T4SS) that allows the organism to kill, in contact-dependent fashion, heterologous bacteria, including wild-type Pseudomonas aeruginosa. Bioinformatic screens based largely on the presence of both a C-terminal consensus sequence and an adjacent gene encoding a cognate immunity protein identified 13 potential antibacterial effectors, most of which were highly conserved among sequenced strains of S. maltophilia. The immunity proteins of two of these proved especially capable of protecting P. aeruginosa and Escherichia coli against attack from the Stenotrophomonas T4SS. In turn, S. maltophilia mutants lacking the putative effectors RS14245 and RS14255 were impaired for killing not only laboratory E. coli but clinical isolates of P. aeruginosa, including ones isolated from the lungs of cystic fibrosis patients. That complemented mutants behaved as wild type did confirmed that RS14245 and RS14255 are required for the bactericidal activity of the S. maltophilia T4SS. Moreover, a mutant lacking both of these proteins was as impaired as a mutant lacking the T4SS apparatus, indicating that RS14245 and RS14255 account for (nearly) all of the bactericidal effects seen. Utilizing an interbacterial protein translocation assay, we determined that RS14245 and RS14255 are bona fide substrates of the T4SS, a result confirmed by examination of mutants lacking both the T4SS and the individual effectors. Delivery of the cloned 14245 protein (alone) into the periplasm resulted in the killing of target bacteria, indicating that this effector, a putative lipase, is both necessary and sufficient for bactericidal activity. IMPORTANCE S. maltophilia is an increasingly important opportunistic pathogen. Inherently resistant to many antibiotics, S. maltophilia is often associated with lung infection, being, among other things, a complicating factor in cystic fibrosis patients. Moreover, it is a common form of coinfection in COVID-19 patients. In these various clinical settings and in natural habitats, S. maltophilia coexists with other pathogens, including P. aeruginosa. Previously, we documented that S. maltophilia possesses a T4SS that kills other bacteria, a notable observation given that most prior work on interbacterial competition has highlighted bactericidal effects of type VI secretion systems. By utilizing approaches ranging from bioinformatics to mutant analysis to protein translocation assays, we have now identified two substrates of the Stenotrophomonas T4SS that largely mediate the killing of pathogenic P. aeruginosa. These results represent a major advance in understanding S. maltophilia, the roles of T4SSs, concepts regarding clinically relevant, interbacterial competition, and activities of bactericidal effectors.

Persistent Bacterial Coinfection of a COVID-19 Patient Caused by a Genetically Adapted Pseudomonas aeruginosa Chronic Colonizer.

Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen which causes chronic infections in immunocompromised patients and leads to high mortality rate. It is identified as a common coinfecting pathogen in COVID-19 patients causing exacerbation of illness. In our hospital, P. aeruginosa is one of the top coinfecting bacteria identified among COVID-19 patients. We collected a strong biofilm-forming P. aeruginosa strain displaying small colony variant morphology from a severe COVID-19 patient. Genomic and transcriptomic sequencing analyses were performed with phenotypic validation to investigate its adaptation in SARS-CoV-2 infected environment. Genomic characterization predicted specific genomic islands highly associated with virulence, transcriptional regulation, and DNA restriction-modification systems. Epigenetic analysis revealed a specific N6-methyl adenine (m6A) methylating pattern including methylation of alginate, flagellar and quorum sensing associated genes. Differential gene expression analysis indicated that this isolate formed excessive biofilm by reducing flagellar formation (7.4 to 1,624.1 folds) and overproducing extracellular matrix components including CdrA (4.4 folds), alginate (5.2 to 29.1 folds) and Pel (4.8-5.5 folds). In summary, we demonstrated that P. aeuginosa clinical isolates with novel epigenetic markers could form excessive biofilm, which might enhance its antibiotic resistance and in vivo colonization in COVID-19 patients.