Mechanistic insights into nanoparticle surface-bacterial membrane interactions in overcoming antibiotic resistance.

Antimicrobial Resistance (AMR) raises a serious concern as it contributes to the global mortality by 5 million deaths per year. The overall impact pertaining to significant membrane changes, through broad spectrum drugs have rendered the bacteria resistant over the years. The economic expenditure due to increasing drug resistance poses a global burden on healthcare community and must be dealt with immediate effect. Nanoparticles (NP) have demonstrated inherent therapeutic potential or can serve as nanocarriers of antibiotics against multidrug resistant (MDR) pathogens. These carriers can mask the antibiotics and help evade the resistance mechanism of the bacteria. The targeted delivery can be fine-tuned through surface functionalization of Nanocarriers using aptamers, antibodies etc. This review covers various molecular mechanisms acquired by resistant bacteria towards membrane modification. Mechanistic insight on 'NP surface-bacterial membrane' interactions are crucial in deciding the role of NP as therapeutic. Finally, we highlight the potential accessible membrane targets for designing smart surface-functionalized nanocarriers which can act as bacteria-targeted robots over the existing clinically available antibiotics. As the bacterial strains around us continue to evolve into resistant versions, nanomedicine can offer promising and alternative tools in overcoming AMR.

Evaluation of indirect sequence-specific magneto-extraction-aided LAMP for fluorescence and electrochemical SARS-CoV-2 nucleic acid detection.

Nucleic acid amplification tests (NAATs) such as quantitative real-time reverse transcriptase PCR (qRT-PCR) or isothermal NAATs (iNAATs) such as loop-mediated isothermal amplification (LAMP) require pure nucleic acid free of any polymerase inhibitors as its substrate. This in turn, warrants the use of spin-column mediated extraction with centralized high-speed centrifuges. Additionally, the utilization of centralized real-time fluorescence readout and TaqMan-like molecular probes in qRT-PCR and real-time LAMP add cost and restrict their deployment. To circumvent these disadvantages, we report a novel sample-to-answer workflow comprising an indirect sequence-specific magneto-extraction (also referred to as magnetocapture, magneto-preconcentration, or magneto-enrichment) for detecting SARS-CoV-2 nucleic acid. It was followed by in situ fluorescence or electrochemical LAMP. After in silico validation of the approach's sequence selectivity against SARS-CoV-2 variants of concern, the comparative performance of indirect and direct magnetocapture in detecting SARS-CoV-2 nucleic acid in the presence of excess host nucleic acid or serum was probed. After proven superior, the sensitivity of the indirect sequence-specific magnetocapture in conjunction with electrochemical LAMP was investigated. In each case, its sensitivity was assessed through the detection of clinically relevant 102 and 103 copies of target nucleic acid. Overall, a highly specific nucleic acid detection method was established that can be accommodated for either centralized real-time SYBR-based fluorescence LAMP or portable electrochemical LAMP.

A batch processed titanium-vanadium oxide nanocomposite based solid-state electrochemical sensor for zeptomolar nucleic acid detection.

Approaching a nucleic acid amplification test (NAAT) based diagnosis of a pathogen from an electrochemistry pathway is a relatively economical, decentralized, and yet highly sensitive route. This work aimed to construct an electrochemical biosensor with a 2-electrode geometry using a transition metal oxide (TMO) based sensing layer. A series of batch-processed TiO2-V2O5 (TVO) nanocomposite-based electrodes were fabricated to probe their electrochemical performance and attain a highly sensitive dual-electrode electrochemical sensor (DEES) compared to pristine V2O5. The XRD analysis of the electrodes confirmed the formation of a nanocomposite, while the XPS analysis correlated the formation of oxygen vacancies with improved electrical conduction measured via EIS and I-V characterization. Furthermore, the work demonstrated the application of the optimized electrode in electrochemical detection of end-point loop-mediated isothermal amplification (LAMP) readout for 101-104 copies (0.1 zeptomoles to 0.1 attomoles) of SARS-CoV-2 RNA dependent RNA polymerase (RdRp) plasmid DNA and in vitro transcribed RNA in an aqueous solution. The device achieved a limit of detection as low as 2.5 and 0.25 copies per µL for plasmid DNA and in vitro transcribed RNA, respectively. The DEES was able to successfully detect in situ LAMP performed on magneto-extracted SARS-CoV-2 plasmid and RNA from (a) an aqueous solution, (b) a sample spiked with excess human genomic DNA, and (c) a serum-spiked sample. The DEES results were then compared with those of real-time fluorescence and commercially available screen-printed electrodes (SPEs).

Nucleic acid extraction from complex biofluid using toothpick-actuated over-the-counter medical-grade cotton.

Nucleic acid amplification technique (NAAT)-assisted detection is the primary intervention for pathogen molecular diagnostics. However, NAATs such as quantitative real-time polymerase chain reaction (qPCR) require prior purification or extraction of target nucleic acid from the sample of interest since the latter often contains polymerase inhibitors. Similarly, genetic disease screening is also reliant on the successful extraction of pure patient genomic DNA from the clinical sample. However, such extraction techniques traditionally utilize spin-column techniques that in turn require centralized high-speed centrifuges. This hinders any potential deployment of qPCR- or PCR-like NAAT methods in resource-constrained settings. The development of instrument-free nucleic acid extraction methods, especially those utilizing readily available materials would be of great interest and benefit to NAAT-mediated molecular diagnosis workflows in resource-constrained settings. In this report, we screened medical-grade cotton, a readily available over-the-counter biomaterial to extract genomic DNA (gDNA) spiked in 30 %, 45 %, and 60 % serum or cell lysate. The extraction was carried out in a completely instrument-free manner using cotton and a sterilized toothpick and was completed in 30 min (with using chaotropic salt) or 10 min (without using chaotropic salt). The quality of the extracted DNA was then probed using PCR followed by agarose gel analysis for preliminary validation of the study. The qPCR experiments then quantitatively established the extraction efficiency (0.3-27 %, depending on serum composition). Besides, percent similarity score obtained from the Sanger sequencing experiments probed the feasibility of extracted DNA towards polymerase amplification with fluorescent nucleotide incorporation. Overall, our method demonstrated that DNA extraction could be performed utilizing toothpick-mounted cotton both with or without using a chaotropic salt, albeit with a difference in the quality of the extracted DNA.

Chromatin immunoprecipitation identifies genes under direct VraSR regulation in Staphylococcus aureus.

Transcriptional profiling of Staphylococcus aureus treated with cell wall-active antibiotics identified the 2-component system, VraSR, as one of the key players in response to antibiotic stress. Although it has been shown that a number of genes are regulated by the VraSR system, it has not been shown which genes are under direct VraSR regulation and which genes are not. In this study, chromatin immunoprecipitation techniques were used to identify the genes which are regulated by the direct interaction of VraR with their promoter regions. The results showed for the first time, that the VraSR mediated regulation of cell wall biosynthesis-associated genes, pbp2, murZ, and sgtB are facilitated by the direct binding of VraR to their respective promoters. Conversely, fmtA, indicated previously to be under VraSR regulation did not exhibit direct regulation by the binding of VraR to its promoter. The VraSR system plays a very important role in antibiotic resistance against cell wall-active antibiotics, and hence, it is essential to understand its complete regulatory mechanism.

The Staphylococcus aureus CsoR regulates both chromosomal and plasmid-encoded copper resistance mechanisms.

Copper is an essential metal which is used as a cofactor in several enzymes and is required for numerous essential biochemical reactions. However, free copper ions can be toxic to cellular systems if the intracellular concentration is not tightly regulated. In this study we show that Staphylococcus aureus copper resistance is not the same in every staphylococcal isolate, but in fact varies considerably between clinical strains. Hyper-copper-resistance was shown to be due to the carriage of an additional plasmid-encoded copper homeostasis mechanism, copBmco. This plasmid can be transferred into the copper-sensitive S. aureus Newman to confer a hyper-copper-resistant phenotype, showing that copper resistance has the potential to spread to other S. aureus strains. This is the first time that plasmid-encoded copper resistance has been reported and shown to be transferable between pathogenic bacteria isolated from humans. A homologue of the Bacillus subtilis and Mycobacterium tuberculosis CsoR regulators was identified in S. aureus. The S. aureus csoR gene is conserved in all sequenced S. aureus genomes and was found to be copper-induced and transcribed along with two downstream genes: a putative copper chaperone (csoZ) and a hypothetical gene. Mutational and complementation studies showed that unlike other homologues, the S. aureus CsoR negatively regulates both chromosomal and plasmid-encoded copper homeostasis mechanisms in response to excess-copper conditions.

Fur is required for the activation of virulence gene expression through the induction of the sae regulatory system in Staphylococcus aureus.

Our previous studies showed that both Sae and Fur are required for the induction of eap and emp expression in low iron. In this study, we show that expression of sae is also iron-regulated, as sae expression is activated by Fur in low iron. We also demonstrate that both Fur and Sae are required for full induction of the oxidative stress response and expression of non-covalently bound surface proteins in low-iron growth conditions. In addition, Sae is required for the induced expression of the important virulence factors isdA and isdB in low iron. Our studies also indicate that Fur is required for the induced expression of the global regulators Agr and Rot in low iron and a number of extracellular virulence factors such as the haemolysins which are also Sae- and Agr-regulated. Hence, we show that Fur is central to a complex regulatory network that is required for the induced expression of a number of important S. aureus virulence determinants in low iron.

Copper stress induces a global stress response in Staphylococcus aureus and represses sae and agr expression and biofilm formation.

Copper is an important cofactor for many enzymes; however, high levels of copper are toxic. Therefore, bacteria must ensure there is sufficient copper for use as a cofactor but, more importantly, must limit free intracellular levels to prevent toxicity. In this study, we have used DNA microarray to identify Staphylococcus aureus copper-responsive genes. Transcriptional profiling of S. aureus SH1000 grown in excess copper identified a number of genes which fall into four groups, suggesting that S. aureus has four main mechanisms for adapting to high levels of environmental copper, as follows: (i) induction of direct copper homeostasis mechanisms; (ii) increased oxidative stress resistance; (iii) expression of the misfolded protein response; and (iv) repression of a number of transporters and global regulators such as Agr and Sae. Our experimental data confirm that resistance to oxidative stress and particularly to H2O2 scavenging is an important S. aureus copper resistance mechanism. Our previous studies have demonstrated that Eap and Emp proteins, which are positively regulated by Agr and Sae, are required for biofilm formation under low-iron growth conditions. Our transcriptional analysis has confirmed that sae, agr, and eap are repressed under high-copper conditions and that biofilm formation is indeed repressed under high-copper conditions. Therefore, our results may provide an explanation for how copper films can prevent biofilm formation on catheters.

Transcriptomic response of Listeria monocytogenes to iron limitation and Fur mutation.

Iron is required by almost all bacteria, but concentrations above physiological levels are toxic. In bacteria, intracellular iron is regulated mostly by the ferric uptake regulator, Fur, or a similar functional protein. Iron limitation results in the regulation of a number of genes, especially those involved in iron uptake. A subset of these genes is the Fur regulon under the control of Fur. In the present study, we have identified Fur- and iron-regulated genes in Listeria monocytogenes by DNA microarray analysis using a fur mutant and its isogenic parent. To identify genes regulated exclusively in response to iron limitation, the whole-genome transcriptional responses to the iron limitation of a fur mutant and its isogenic parent were compared. Fur-regulated genes were identified by comparing the transcriptional profile of the parent with the transcriptional profile of the isogenic fur mutant. Our studies have identified genes regulated exclusively in response to iron and those that are negatively regulated by Fur. We have identified at least 14 genes that were negatively regulated directly by Fur. Under iron-limited conditions, these genes were upregulated, while the expression of fur was found to be downregulated. To further investigate the regulation of fur in response to iron, an ectopic fur promoter-lacZ transcriptional fusion strain was constructed, and its isogenic fur and perR mutant derivatives were generated in L. monocytogenes 10403S. Analysis of the iron limitation of the perR mutant indicated that the regulation of genes under the negative control of Fur was significantly inhibited. Our results indicate that Fur and PerR proteins negatively regulate fur and that under iron-limited conditions, PerR is required for the negative regulation of genes controlled by Fur.