Activity-Based Protein Profiling in Methicillin-Resistant Staphylococcus aureus Reveals the Broad Reactivity of a Carmofur-Derived Probe.

Activity-based protein profiling is a powerful chemoproteomic technique to detect active enzymes and identify targets and off-targets of drugs. Here, we report the use of carmofur- and activity-based probes to identify biologically relevant enzymes in the bacterial pathogen Staphylococcus aureus. Carmofur is an anti-neoplastic prodrug of 5-fluorouracil and also has antimicrobial and anti-biofilm activity. Carmofur probes were originally designed to target human acid ceramidase, a member of the NTN hydrolase family with an active-site cysteine nucleophile. Here, we first profiled the targets of a fluorescent carmofur probe in live S. aureus under biofilm-promoting conditions and in liquid culture, before proceeding to target identification by liquid chromatography/mass spectrometry. Treatment with a carmofur-biotin probe led to enrichment of 20 enzymes from diverse families awaiting further characterization, including the NTN hydrolase-related IMP cyclohydrolase PurH. However, the probe preferentially labeled serine hydrolases, thus displaying a reactivity profile similar to that of carbamates. Our results suggest that the electrophilic N-carbamoyl-5-fluorouracil scaffold could potentially be optimized to achieve selectivity towards diverse enzyme families. The observed promiscuous reactivity profile suggests that the clinical use of carmofur presumably leads to inactivation of a number human and microbial enzymes, which could lead to side effects and/or contribute to therapeutic efficacy.

Associations between antibiotic resistance and bacteriophage resistance phenotypes in laboratory and clinical strains of Salmonella enterica subsp. enterica serovar Typhimurium.

Bacteriophages have received great attention as an alternative over antibiotics due to the host specificity. Therefore, this study was designed to evaluate the associations between bacteriophage-insensitive (BI) and antibiotic-resistant mutants of Salmonella Typhimurium strains. Bacteriophage-sensitive (BS) Salmonella enterica serovar Typhimurium ATCC 19585 (BSSTWT), ciprofloxacin-induced S. Typhimurium ATCC 19585 (BSSTCIP), S. Typhimurium KCCM 40253 (BSSTLAB), and clinically isolated multidrug-resistant S. Typhimurium CCARM 8009 (BSSTMDR) were used to induce the bacteriophage-insensitive mutants (BISTWT, BISTCIP, BISTLAB, and BISTMDR), which were characterized by measuring mutant frequency lysogenic induction, phage adsorption, antibiotic susceptibility, and differential gene expression. The numbers of BSSTWT, BSSTCIP, and BSSTLAB were reduced by P22 (>3 log), while the least lytic activity was observed for BSSTMDR, suggesting alteration in bacteriophage-binding receptors on the surface of multidrug-resistant strain. BSSTWT treated with P22 showed the large variation in the cell state (CV>40%) and highest mutant frequency (62%), followed by 25% for BSSTCIP. The least similarities between BSSTWT and BISTWT were observed for P22 and PBST-13 (<12%). The relative expression levels of bacteriophage-binding receptor-related genes (btuB, fhuA, fliK, fljB, ompC, ompF, rfaL, and tolC) were decreased in BISTCIP and BISTMDR. These results indicate that the bacteriophage resistance is highly associated with the antibiotic resistance. The findings in this study could pave the way for the application of bacteriophages as an alternative to control antibiotic-resistant bacteria.

From Differential Stains to Next Generation Physiology: Chemical Probes to Visualize Bacterial Cell Structure and Physiology.

Chemical probes have been instrumental in microbiology since its birth as a discipline in the 19th century when chemical dyes were used to visualize structural features of bacterial cells for the first time. In this review article we will illustrate the evolving design of chemical probes in modern chemical biology and their diverse applications in bacterial imaging and phenotypic analysis. We will introduce and discuss a variety of different probe types including fluorogenic substrates and activity-based probes that visualize metabolic and specific enzyme activities, metabolic labeling strategies to visualize structural features of bacterial cells, antibiotic-based probes as well as fluorescent conjugates to probe biomolecular uptake pathways.

Assessment of antibiotic resistance in bacteriophage-insensitive Klebsiella pneumoniae.

This study was design to evaluate the physiological properties of bacteriophage-insensitive Klebsiella pneumoniae (BIKP) mutants in association with the antibiotic cross-resistance, ß-lactamase activity, and gene expression. Klebsiella pneumoniae ATCC 23357(KPWT), ciprofloxacin-induced antibiotic-resistant K. pneumoniae ATCC 23357 (KPCIP), and clinically isolated antibiotic-resistant K. pneumoniae 10263 (KPCLI) were used to isolate BIKP mutants against KPB1, PBKP02, PBKP21, PBKP29, PBKP33, and PBKP35. PBKP35-induced mutants, including bacteriophage-insensitive K. pneumoniae ATCC 23357 (BIKPWT), ciprofloxacin-induced K. pneumoniae ATCC 23357 (BIKPCIP), and clinically isolated antibiotic-resistant K. pneumoniae CCARM 10263 (BIKPCLI). BIKPWT, BIKPCIP, and BIKPCLI were resistant to Klebsiella bacteriophages, KPB1, PBKP02, PBKP21, PBKP29, and PBKP33. The antibiotic cross-resistance to cefotaxime, cephalothin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, levofloxacin, and nalidixic acid was observed in BIKPWT. The relative expression levels of vagC was increased by more than 8-folds in BIKPWT, corresponding to the increased ß-lactamase activity. The aac(6')-Ib-cr was overexpressed in BIKP mutants, responsible for aminoglycoside and quinolone resistance. The phage-resistant mutants decreased the antibiotic susceptibilities in association with ß-lactamase activity and antibiotic resistance-related gene expression. The results pointed out the cross-resistance of BIKP mutants to antibiotics, which might be considered when applying for the therapeutic use of bacteriophage.

Assessment of the alteration in phage adsorption rates of antibiotic-resistant Salmonella typhimurium.

This study was designed to evaluate the phage-binding receptors on the surface of antibiotic-sensitive Salmonella typhimurium (ASST) and antibiotic-resistant S. typhimurium (ARST). The antibiotic susceptibilities of plasmid-cured ASST and ARST were evaluated against ampicillin, cephalothin, ciprofloxacin, kanamycin, penicillin, and tetracycline. The capsular polysaccharides (CPSs) and lipopolysaccharides (LPSs) were quantified using carbazole assay and HPLC, respectively. The amounts of CPSs and LPSs in ARST were decreased from 108 to 62 µg/ml and 284-111 ng/ml, respectively, after plasmid curing. The adsorption rates of P22, PBST10, and PBST13 to plasmid-uncured and plasmid-cured ASST and ARST were decreased after proteinase K and periodate treatments. The highest reduction in phage adsorption rate was observed for P22 to the plasmid-cured ARST treated with periodate (71%). The relative expression levels of btuB, fhuA, and rfaL were decreased by more than twofold in the plasmid-cured ASST, corresponding to the decrease in the adsorption rates of P22 and PBST10. The plasmid-cured ARST lost the ability to express the ß-lactamase gene, which was related to the loss of resistance to ampicillin, cephalothin, kanamycin, penicillin, and tetracycline. The results provide valuable insights into understanding the interaction between phage and antibiotic-resistant bacteria.

Proteomics-based discrimination of differentially expressed proteins in antibiotic-sensitive and antibiotic-resistant Salmonella Typhimurium, Klebsiella pneumoniae, and Staphylococcus aureus.

This study was designed to compare the differentially expressed proteins between antibiotic-sensitive and antibiotic-resistant Salmonella Typhimurium, Klebsiella pneumonia, and Staphylococcus aureus. The susceptibilities of wild-type (WT), ciprofloxacin (CIP) and/or oxacillin (OXA)-induced, and clinically isolated resistant (CCARM) S. Typhimurium (STWT, STCIP, and STCCARM), K. pneumoniae (KPWT, KPCIP, and KPCCARM), and S. aureus (SAWT, SACIP, SAOXA, and SACCARM) to antibiotics were determined using broth microdilution assay. STCIP was highly resistant to piperacillin (MIC > 512 µg/ml), KPCIP was resistant to chloramphenicol (128 µg/ml) and norfloxacin (16 µg/ml), SACIP was resistant to fluoroquinolones (32 µg/ml), and SAOXA was resistant to ceftriaxone (32 µg/ml). The protein profiles of antibiotic-sensitive and antibiotic-resistant strains were determined using 2-DE analysis followed by LC-MS/MS. The commonly expressed proteins of STWT-STCIP, STWT-STCCARM, KPWT-KPCIP, KPWT-KPCCARM, SAWT-SACIP, SAWT-SAOXA, and SAWT-SACCARM were 763, 677, 677, 469, 261, 259, and 226, respectively. The unique protein spots were observed 57 (6.5%), 80 (11.5%), and 68 (13.9%), respectively, for STCCARM, KPCCARM, and SACCARM. The highly up-regulated protein, PrsA (10-fold), was observed in STCIP resistant to ciprofloxacin (128-fold), levofloxacin (32-fold), norfloxacin (64-fold), and piperacillin (> 16-fold). The up-regulated proteins (YadC, FimA, and RplB) in KPCIP resistant to chloramphenicol (> 32-fold), ciprofloxacin (32-fold), levofloxacin (6-fold), norfloxacin (128-fold), and sparfloxacin (64-fold). AcrB and RpoB were up-regulated in SACCARM resistant to multiple antibiotics. The differentially expressed proteins were related to the antibiotic resistance of STWT, STCIP, STCCARM, KPWT, KPCIP, KPCCARM, SAWT, SACIP, SAOXA, and SACCARM. The resistance-associated proteins could be useful biomarkers for detecting antibiotic-resistant pathogens.

Vanda roxburghii chloroform extract as a potential source of polyphenols with antioxidant and cholinesterase inhibitory activities: identification of a strong phenolic antioxidant.

BACKGROUND: Alzheimer's disease (AD) is a progressively developing neurodegenerative disorder of the brain in the elderly people. Vanda roxburghii Rbr. root has been used traditionally in Bangladesh as tonic to brain and in the treatment of nervous system disorders including AD. Therefore, we aimed to investigate the cholinesterase inhibitory activities and antioxidant properties of the extracts from V. roxburghii. METHODS: The crude methanol extract from the roots of plant was sequentially fractionated with petroleum ether, chloroform, ethylacetate and water to yield their corresponding extracts. The extracts were assessed for acetylcholinesterase and butyrylcholinesterase inhibitory activity by modified Ellman method and antioxidant property by several assays including ferric reducing antioxidant power, scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and hydroxyl radical, and inhibition of lipid peroxidation. Endogenous substances in the extracts were analyzed by the standard phytochemical methods and active compound was isolated by the chromatographic methods. RESULTS: Chloroform extract was shown to demonstrate strong ferric-reducing antioxidant power and scavenging activity against DPPH and hydroxyl free radicals when compared with the other extracts and the reference standard catechin. The antioxidant effect was further verified by inhibition of lipid peroxidation in rat brain homogenates. Likewise, the chloroform extract exhibited the highest inhibition against both the acetylcholinesterase and butyrylcholinesterase enzymes with IC50 values of 221.13 and 82.51 µg/ml, respectively. Phytochemical screening revealed a large amount of phenolics and flavonoids in the chloroform extract. Bioactivity guided separation techniques led to the isolation of a strong antioxidant from the chloroform extract and its structure was determined as gigantol on the basis of spectral studies. CONCLUSION: These results suggest that the chloroform extract of V. roxburghii, possibly due to its phenolic compounds, exert potential antioxidant and cholinesterase inhibitory activities, which may be useful in the treatment of AD.

An Internet-of-Disease System for COVID-19 Testing Using Saliva by an AI-Controlled Microfluidic ELISA Device.

Throughout coronavirus disease (COVID-19) outbreaks, the centers for disease control and prevention (CDCP) of a country require monitoring of particular territories to provide public health guidance. In this work, the Internet of Diseases (IoD) is suggested for continuous real-time monitoring of infectious diseases for public health. Because converging information and communication technologies (ICTs) with point-of-care (POC) devices to enable the IoD for continuous real-time health monitoring and processing of clinical records are crucial, an IoD platform associating a lab-on-a-chip (LOC) device to diagnose severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) from oropharyngeal saliva samples have been developed and uploaded the resulted diagnostic data into a cloud-based system to be connected with CDCP. Moreover, a choropleth IoD map to visualize provincial infection rate is proposed along with the IoD platform. The developed platform is applied for the quantification of SARS-CoV-2 N-protein antigen with a LOD as low as 0.013 ng mL-1 and the infection rate of various provinces is projected with the IoD map successfully. Thus, the proposed IoD system has the potential to become an imperative tool for the disease control and prevention centers to restrain COVID-19 outbreaks by identifying the severity of particular regions.

Exploring the Journey of Zinc Oxide Nanoparticles (ZnO-NPs) toward Biomedical Applications.

The field of nanotechnology is concerned with the creation and application of materials having a nanoscale spatial dimensioning. Having a considerable surface area to volume ratio, nanoparticles have particularly unique properties. Several chemical and physical strategies have been used to prepare zinc oxide nanoparticles (ZnO-NPs). Still, biological methods using green or natural routes in various underlying substances (e.g., plant extracts, enzymes, and microorganisms) can be more environmentally friendly and cost-effective than chemical and/or physical methods in the long run. ZnO-NPs are now being studied as antibacterial agents in nanoscale and microscale formulations. The purpose of this study is to analyze the prevalent traditional method of generating ZnO-NPs, as well as its harmful side effects, and how it might be addressed utilizing an eco-friendly green approach. The study's primary focus is on the potential biomedical applications of green synthesized ZnO-NPs. Biocompatibility and biomedical qualities have been improved in green-synthesized ZnO-NPs over their traditionally produced counterparts, making them excellent antibacterial and cancer-fighting drugs. Additionally, these ZnO-NPs are beneficial when combined with the healing processes of wounds and biosensing components to trace small portions of biomarkers linked with various disorders. It has also been discovered that ZnO-NPs can distribute and sense drugs. Green-synthesized ZnO-NPs are compared to traditionally synthesized ones in this review, which shows that they have outstanding potential as a potent biological agent, as well as related hazardous properties.

Exogenous putrescine attenuates the negative impact of drought stress by modulating physio-biochemical traits and gene expression in sugar beet (Beta vulgaris L.).

Drought tolerance is a complex trait controlled by many metabolic pathways and genes and identifying a solution to increase the resilience of plants to drought stress is one of the grand challenges in plant biology. This study provided compelling evidence of increased drought stress tolerance in two sugar beet genotypes when treated with exogenous putrescine (Put) at the seedling stage. Morpho-physiological and biochemical traits and gene expression were assessed in thirty-day-old sugar beet seedlings subjected to drought stress with or without Put (0.3, 0.6, and 0.9 mM) application. Sugar beet plants exposed to drought stress exhibited a significant decline in growth and development as evidenced by root and shoot growth characteristics, photosynthetic pigments, antioxidant enzyme activities, and gene expression. Drought stress resulted in a sharp increase in hydrogen peroxide (H2O2) (89.4 and 118% in SBT-010 and BSRI Sugar beet 2, respectively) and malondialdehyde (MDA) (35.6 and 27.1% in SBT-010 and BSRI Sugar beet 2, respectively). These changes were strongly linked to growth retardation as evidenced by principal component analysis (PCA) and heatmap clustering. Importantly, Put-sprayed plants suffered from less oxidative stress as indicated by lower H2O2 and MDA accumulation. They better regulated the physiological processes supporting growth, dry matter accumulation, photosynthetic pigmentation and gas exchange, relative water content; modulated biochemical changes including proline, total soluble carbohydrate, total soluble sugar, and ascorbic acid; and enhanced the activities of antioxidant enzymes and gene expression. PCA results strongly suggested that Put conferred drought tolerance mostly by enhancing antioxidant enzymes activities that regulated homeostasis of reactive oxygen species. These findings collectively provide an important illustration of the use of Put in modulating drought tolerance in sugar beet plants.

The Role of Bacterial Membrane Vesicles in the Dissemination of Antibiotic Resistance and as Promising Carriers for Therapeutic Agent Delivery.

The rapid emergence and spread of antibiotic-resistant bacteria continues to be an issue difficult to deal with, especially in the clinical, animal husbandry, and food fields. The occurrence of multidrug-resistant bacteria renders treatment with antibiotics ineffective. Therefore, the development of new therapeutic methods is a worthwhile research endeavor in treating infections caused by antibiotic-resistant bacteria. Recently, bacterial membrane vesicles (BMVs) have been investigated as a possible approach to drug delivery and vaccine development. The BMVs are released by both pathogenic and non-pathogenic Gram-positive and Gram-negative bacteria, containing various components originating from the cytoplasm and the cell envelope. The BMVs are able to transform bacteria with genes that encode enzymes such as proteases, glycosidases, and peptidases, resulting in the enhanced antibiotic resistance in bacteria. The BMVs can increase the resistance of bacteria to antibiotics. However, the biogenesis and functions of BMVs are not fully understood in association with the bacterial pathogenesis. Therefore, this review aims to discuss BMV-associated antibiotic resistance and BMV-based therapeutic interventions.

Development of de novo resistance in Salmonella Typhimurium treated with antibiotic combinations.

This study was designed to evaluate the evolution of antibiotic resistance in Salmonella enterica serovar Typhimurium treated with the combination of antibiotics. The experimental evolution of antibiotic resistance of S. Typhimurium was evaluated either under single antibiotic (kanamycin, KAN; penicillin, PEN; erythromycin, ERY) or in combination of two antibiotics (KAN + PEN or KAN + ERY) as measured by fractional inhibitory concentrations (FICs), stepwise resistance selection, cross-resistance evaluation, resistance fitness and relative gene expression. KAN + PEN and KAN + ERY showed the synergistic effect against S. Typhimurium (FIC index < 0.5). KAN + ERY delayed the induction of de novo mutations in S. Typhimurium. The cross-resistance of S. Typhimurium to all antibiotics except ERY and tetracycline was observed in KAN and PEN alone. The fitness cost was lower in single antibiotic treatments than combinations. The highest relative fitness was 0.91 in PEN, followed by KAN (0.84) and ERY (0.78), indicating the low fitness costs in single antibiotic treatments. The overexpression of efflux pump-related genes (acrA and acrB), outer membrane-related gene (ompC) and adherence-related gene (csgD) were observed in the single antibiotic treatments. Our results suggest that KAN + PEN and KAN + ERY could be used as a potential therapeutic treatment by decreasing the evolution of antibiotic resistance in S. Typhimurium and reusing conventional antibiotics.

Variability in the Adaptive Response of Antibiotic-Resistant Salmonella Typhimurium to Environmental Stresses.

This study was designed to evaluate the resistance phenotype and genotype of wild type (WT)-, cefotaxime (CET)-, and ciprofloxacin (CIP)-induced Salmonella Typhimurium ATCC 19585, CIP-resistant Salmonella Typhimurium ATCC 19585, Salmonella Typhimurium CCARM 8009, and Salmonella Typhimurium KCCM 40253 before and after exposure to pH 4.5, 4% NaCl, and heat at 42°C. The susceptibilities of WT Salmonella Typhimurium ATCC 19585 and WT Salmonella Typhimurium KCCM 40253 to all antibiotics tested in this study were decreased after CET and CIP induction with the exception with kanamycin, meropenem, and polymyxin B. The highest ß-lactamase activities were 2.8 and 3.3 nmol/(min·mL), respectively, at the WT- and CET-induced Salmonella Typhimurium CCARM 8009. FT-IR spectra were found to be dominant at the region from 1,700 to 1,500 cm-1 corresponding to proteins such as amides I, II, and III. The relative expression levels of efflux pump-related genes (acrA, acrB, and TolC), porin-related gene (ompC), virulence-related gene (stn), adhesion-related gene (fimA), and stress-induced alternative sigma factor (rpoS) varied in the antibiotic resistance and stress exposure. This study provides useful information for understanding the antibiotic resistance profile, physicochemical property, and gene expression pattern in Salmonella Typhimurium in association with the induction of antibiotic resistance and exposure to environmental stresses.

Bacteriophages as Potential Tools for Detection and Control of Salmonella spp. in Food Systems.

The global problem of antibiotic resistance in bacteria is quickly developing in most antibiotics used in hospitals and livestock. Recently, the infections with multi-drug resistant (MDR) bacteria become a major cause of death worldwide. Current antibiotics are not very effective in treating MDR Salmonella infections, which have become a public health threat. Therefore, novel approaches are needed to rapidly detect and effectively control antibiotic-resistant pathogens. Bacteriophages (phages) have seen renewed attention for satisfying those requirements due to their host-specific properties. Therefore, this review aims to discuss the possibility of using phages as a detection tool for recognizing bacterial cell surface receptors and an alternative approach for controlling antibiotic-resistant pathogens in food systems.

Characterization of ß-lactamase- and efflux pump-mediated multiple antibiotic resistance in Salmonella Typhimurium.

This study aimed to assess the ß-lactamase- and efflux pump-mediated antibiotic resistance in Salmonella Typhimurium (WT-ST), ciprofloxacin-induced antibiotic-resistant S. Typhimurium (CI-ST), and clinically-acquired antibiotic-resistant S. Typhimurium (CA-ST). The ß-lactamase activities were significantly increased up to 63 µmol/min/mL in CA-ST and 24 µmol/min/mL in CI-ST when compared to WT-ST (13 µmol/min/mL). The highest efflux pump activity was observed in CI-ST and CA-ST, showing more than 45%. The antibiotic susceptibilities of WT-ST, CI-ST, and CA-ST were increased in the presence of ß-lactamase and efflux pump inhibitors. CA-ST showed the highest activity in AcrD, MdtABC, EmrAB, MdtK, and MacAB efflux pumps. The repressed ompF were responsible for the decreased susceptibility of CA-ST to ampicillin (MIC > 512 µg/mL). This study would provide useful information for better understating of the development of multidrug resistance in association with ß-lactamase and efflux pump activities and designing new antibiotic chemotherapy in combination with inhibitors.

Combined effect of bacteriophage and antibiotic on the inhibition of the development of antibiotic resistance in Salmonella typhimurium.

This study was designed to evaluate the combined effects of bacteriophage and antibiotic on the reduction of the development of antibiotic-resistance in Salmonella typhimurium LT2. The susceptibilities of S. typhimurium to ciprofloxacin and erythromycin were increased when treated with bacteriophages, showing more than 10% increase in clear zone sizes and greater than twofold decrease in minimum inhibitory concentration values. The growth of S. typhimurium was effectively inhibited by the combination of bacteriophage P22 and ciprofloxacin. The combination treatment effectively reduced the development of antibiotic resistance in S. typhimurium. The relative expression levels of efflux pump-related genes (acrA, acrB, and tolC) and outer membrane-related genes (ompC, ompD, and ompF) were decreased at all treatments. This study provides useful information for designing new antibiotic therapy to control antibiotic-resistant bacteria.