Intact Bacterial Cells Research Articles

Counting mRNA Copies in Intact Bacterial Cells by Fluctuation Localization Imaging-Based Fluorescence In Situ Hybridization (fliFISH).

A method for measuring mRNA copies in intact bacterial cells by fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) is presented. Unlike conventional single-molecule FISH, where the presence of a transcript is determined by fluorescence intensity, fliFISH relies on On-Off duty cycles of photo-switching dyes to set a predetermined threshold for distinguishing true signals from background noise. The method provides a quantitative approach for detecting and counting true mRNA copies and rejecting false signals with high accuracy.

Synthesis and Matrix Properties of α-Cyano-5-phenyl-2,4-pentadienic Acid (CPPA) for Intact Proteins Analysis by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

The effectiveness of a synthesized matrix, α-cyano-5-phenyl-2,4-pentadienic acid (CPPA), for protein analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in complex samples such as foodstuff and bacterial extracts, is demonstrated. Ultraviolet (UV) absorption along with laser desorption/ionization mass spectrometry (LDI-MS) experiments were systematically conducted in positive ion mode under standard Nd:YLF laser excitation with the aim of characterizing the matrix in terms of wavelength absorption and proton affinity. Besides, the results for standard proteins revealed that CPPA significantly enhanced the protein signals, reduced the spot-to-spot variability and increased the spot homogeneity. The CPPA matrix was successful employed to investigate intact microorganisms, milk and seed extracts for protein profiling. Compared to conventional matrices such as sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA) and 4-chloro-α-cyanocinnamic acid (CClCA), CPPA exhibited better signal-to-noise (S/N) ratios and a uniform response for most examined proteins occurring in milk, hazelnut and in intact bacterial cells of E. coli. These findings not only provide a reactive proton transfer MALDI matrix with excellent reproducibility and sensitivity, but also contribute to extending the battery of useful matrices for intact protein analysis.

Antibacterial effect of a brominated self-etch adhesive on carious dentin - An in vivo study

ObjectivesThis in vivo study compared the antibacterial effect of a self-etch adhesive with and without the brominated monomer 12-methacryloyloxydodecyl-pyridinium bromide (MDPB) on carious dentin after selective caries removal. Methods10 patients showing deep primary carious lesions at two posterior teeth without pulpal symptoms were included. At visit I, carious tissue was selectively removed and carious dentin was sampled with a sterile roundbur (Komet No. 18). One cavity was restored with composite (SDR, Ceram X; DENTSPLY DeTrey) using an MDPB-containing self-etch adhesive (Clearfil Protect Bond, Kuraray Noritake; PB). The other restoration served as a control (Clearfil SE Bond II, Kuraray Noritake; SE). At visit II after 8 weeks, carious dentin was sampled again. Bacterial growth in carious dentin was differentiated using microbial cultivation. Bacterial DNA from intact cells and cell-free DNA were quantified using 16S rRNA gene-based real-time PCR and the microbial community composition was analyzed by amplicon deep-sequencing. Wilcoxon test was applied for statistical analysis. ResultsBoth treatments showed a decrease of intact bacterial cells in carious dentin at visit II compared to visit I (PB: visit I: 1.1*106, visit II: 1.7*105 (p = 0.03); SE: visit I: 1.1*107, visit II = 2.4*105 (p = 0.002)). No statistically significant reduction of cell-free bacterial DNA was detected (PB: visit I: 6.1*105, visit II: 1.6*105 (p = 0.08); SE: visit I: 5.3*105, visit II: 2.9*105 (p = 0.10)). The decrease of intact cell-derived (p = 0.371) and cell-free DNA (p = 0.455) did not differ significantly between PB and SE. Lactobacillus was most abundant within the microbial community at both visits. Alpha-diversity was not affected by treatment and samples showed high intra- and interindividual diversity. Conclusion and clinical significanceBoth self-etch adhesives have an antibacterial effect due to a decrease of bacterial DNA after selective caries removal. However, the results do not reveal any additional antibacterial effect by MDPB.The study is registered with the German Clinical Trials Register (DRKS00011532).

Distribution of extracellular and intracellular antibiotic resistance genes in sludge fractionated in terms of settleability

The widespread proliferation of antibiotic resistance genes (ARGs) is a serious environmental and human health issue. Wastewater treatment facilities (WWTFs) are potential sources to spread ARGs to natural environment, for which, the presence state of ARGs in the sludge, as extracellular ones (eARGs) or intracellular ones (iARGs), along with the sludge settleability, are very important factors. The sludge settleability is closely associated with its floc size and density, bacterial activity, and the proportion of intact/damaged bacterial cells that aggregate together to form flocs for separation in the sedimentation process. It is reasonable to hypothesize that the distribution of eARGs and iARGs may differ with the sludge fractions of different settleability, a topic of great academic and practical significance requiring clarification. In this study, sludge samples from the aerobic contact tank of six household WWTFs were fractionated into fractions with different settling velocities: sludge of low settleability (LS), medium settleability (MS) and high settleability (HS); and the distribution of eARGs and iARGs in the obtained fractions for the widely detected tet G, tet M and sul 1 in water environment was evaluated based on the PMA-qPCR method, together with the evaluation for the well reported mobile genomic element intl 1 and total bacterial 16S rDNA. For the LS fractions, which contained more damaged bacterial cells, the distribution percentages of eARGs were generally higher than those of iARGs. For the HS fractions, which contained flocs with larger sizes formed by both intact and damaged bacterial cells, the relative abundances of ARGs and intl 1 were found apparently lower even if the presence percentages of eARGs were comparatively higher. It is thus inferable that sludge fractions of LS may possess higher transfer potential for ARGs and enhancing their settleability through optimization of the operation conditions is important for mitigating the proliferation of ARGs.

Bacterial viability and diversity in a landscape lake replenished with reclaimed water: a case study in Xi'an, China.

To understand the characteristics of bacterial viability and diversity in landscape waters replenished with reclaimed water, the typical landscape lake using reclaimed water was investigated in this study. Samples were collected from a reclaimed water inlet (P1), a reclaimed water distribution outlet (P2), and a landscape lake replenished by reclaimed water (P3). By means of measuring adenosine triphosphate (ATP), flow cytometry (FCM), and 16S rRNA gene high-throughput sequencing, the bacterial viability and diversity in reclaimed water distribution system and landscape lake were illustrated. The bacterial ATP contents at P1, P2, and P3 were 3.55 ± 1.79ng/L, 3.31 ± 1.43ng/L, and 18.97 ± 6.39μg/L, and the intact bacterial cell concentrations were 5.91 ± 0.52 × 104 cells/mL, 7.95 ± 2.58 × 104 cells/mL, and 5.65 ± 2.10 × 106 cells/mL, respectively. These results indicated a significant increase of bacterial viability in the landscape lake. The Shannon diversity index of 6.535, 7.05, and 6.886 at P1, P2, and P3, respectively, demonstrated no notable change of bacterial diversity from reclaimed water distribution system to landscape lake. However, the relative abundance of Pseudomonas sp. at P3 was significantly higher than that at P1. These findings indicated that viable but non-culturable (VBNC) bacteria could be revived in the landscape lake. The bacterial viability during reclaimed water reuse should deserve special attention.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Utilization of Mn‐Doped ZnSe/ZnS Core/Shell Quantum Dots for Rapid Detection of Escherichia coli O157:H7 and Methicillin‐Resistant Staphylococcus aureus

In this study, Mn‐doped ZnSe/ZnS core/shell quantum dots (CSQDs) were synthesized in aqueous solution using polyethylene glycol as a surface stabilizer and successfully applied in the detection of Escherichia coli O157:H7 and methicillin‐resistant Staphylococcus aureus (MRSA) for the first time. The CSQDs were conjugated with anti‐E. coli antibody and anti‐MRSA antibody via protein A supported by 1‐ethyl‐3‐(‐3‐dimethylaminopropyl)carbodiimide hydrochloride for fluorescent labeling of the intact bacterial cells. The detection was performed for the bacterial strains cultivated in Luria‐Bertani liquid medium. The obtained results indicate that E. coli O157:H7 and MRSA can be detected within 30 min at a high sensitivity of 101 CFU/mL. This labeling method based on the highly fluorescent CSQDs may have great potential for use in the food industry to check and prevent outbreaks of foodborne illness.

Plasmin inhibition by bacterial serpin: Implications in gum disease

Tannerella forsythia is a periodontopathogen that expresses miropin, a protease inhibitor in the serpin superfamily. In this study, we show that miropin is also a specific and efficient inhibitor of plasmin; thus, it represents the first proteinaceous plasmin inhibitor of prokaryotic origin described to date. Miropin inhibits plasmin through the formation of a stable covalent complex triggered by cleavage of the Lys368-Thr369 (P2-P1) reactive site bond with a stoichiometry of inhibition of 3.8 and an association rate constant (kass) of 3.3×105M-1s-1. The inhibition of the fibrinolytic activity of plasmin was nearly as effective as that exerted by α2-antiplasmin. Miropin also acted in vivo by reducing blood loss in a mice tail bleeding assay. Importantly, intact T. forsythia cells or outer membrane vesicles, both of which carry surface-associated miropin, strongly inhibited plasmin. In intact bacterial cells, the antiplasmin activity of miropin protects envelope proteins from plasmin-mediated degradation. In summary, in the environment of periodontal pockets, which are bathed in gingival crevicular fluid consisting of 70% of blood plasma, an abundance of T. forsythia in the bacterial biofilm can cause local inhibition of fibrinolysis, which could have possible deleterious effects on the tooth-supporting structures of the periodontium.

Purple Bacteria and 3D Redox Hydrogels for Bioinspired Photo-bioelectrocatalysis.

A major challenge for the implementation of intact bacterial cells in photo-bioelectrochemical systems remains the hindered extracellular electron transfer. This study focuses on purple bacteria, photosynthetic microorganisms particularly interesting for the development of bioelectrochemical systems because of their versatile metabolisms. Although soluble monomeric redox mediators have been proven as effective systems for electron transfer mediation, their application in the field is not preferable owing to their toxicity and unwanted release into the environment. An abiotic/biotic photoanode is reported in which a bioinspired redox mediating system is implemented in a 3D geometry allowing to "electrically wire" intact bacterial cells. The 3D photoanode decreased the overpotential required for harvesting photoexcited electrons, operating at +0.073 V versus the saturated calomel electrode (SCE). Accordingly, the overpotential was significantly reduced compared with a pioneering Os-redox polymer reported in literature, which required operation at +0.303 V versus SCE. These results provide the basis for further development of bio-photoanodes for light-powered biosensing and power generation.

An in vitro protocol for rapidly assessing the effects of antimicrobial compounds on the unculturable bacterial plant pathogen, Candidatus Liberibacter asiaticus

BackgroundMost bacteria are not culturable, but can be identified through molecular methods such as metagenomics studies. Due to specific metabolic requirements and symbiotic relationships, these bacteria cannot survive on typical laboratory media. Many economically and medically important bacteria are unculturable; including phloem-limited plant pathogens like Candidatus Liberibacter asiaticus (CLas). CLas is the most impactful pathogen on citrus production, is vectored by the Asian citrus psyllid (ACP, Diaphorina citri), and lacks an effective treatment or resistant cultivars. Research into CLas pathogenicity and therapy has been hindered by the lack of persistent pure cultures. Work to date has been mostly limited to in planta studies that are time and resource intensive.ResultsWe developed and optimized an in vitro protocol to quickly test the effectiveness of potential therapeutic agents against CLas. The assay uses intact bacterial cells contained in homogenized tissue from CLas-infected ACP and a propidium monoazide (PMA) assay to measure antimicrobial activity. The applicability of PMA was evaluated; with the ability to differentiate between intact and disrupted CLas cells confirmed using multiple bactericidal treatments. We identified light activation conditions to prevent PCR interference and identified a suitable positive control for nearly complete CLas disruption (0.1% Triton-X 100). Isolation buffer components were optimized with 72 mM salt mixture, 1 mM phosphate buffer and 1% glycerol serving to minimize unwanted interactions with treatment and PMA chemistries and to maximize recovery of intact CLas cells. The mature protocol was used to compare a panel of peptides already under study for potential CLas targeting bactericidal activity and identify which were most effective.ConclusionThis psyllid homogenate assay allows for a quick assessment of potential CLas-disrupting peptides. Comparison within a uniform isolate largely eliminates experimental error arising from variation in CLas titer between and within individual host organisms. Use of an intact vs. disrupted assay permits direct assessment of potential therapeutic compounds without generating pure cultures or conducting extensive in planta or field studies.

Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis

Porphyromonas gingivalis uses a type IX secretion system (T9SS) to deliver more than 30 proteins to the bacterial surface using a conserved C-terminal domain (CTD) as an outer membrane translocation signal. On the surface, the CTD is cleaved and an anionic lipopolysaccharide (A-PLS) is attached by PorU sortase. Among T9SS cargo proteins are cysteine proteases, gingipains, which are secreted as inactive zymogens requiring removal of an inhibiting N-terminal prodomain (PD) for activation. Here, we have shown that the gingipain proRgpB isolated from the periplasm of a T9SS-deficient P. gingivalis strain was stable and did not undergo autocatalytic activation. Addition of purified, active RgpA or RgpB, but not Lys-specific Kgp, efficiently cleaved the PD of proRgpB but catalytic activity remained inhibited because of inhibition of the catalytic domain in trans by the PD. In contrast, active RgpB was generated from the zymogen, although at a slow rate, by gingipain-null P. gingivalis lysate or intact bacterial cell suspension. This activation was dependent on the presence of the PorU sortase. Interestingly, maturation of proRgpB with the catalytic cysteine residues mutated to Ala expressed in the ΔRgpA mutant strain was indistinguishable from that in the parental strain. Cumulatively, this suggests that PorU not only has sortase activity but is also engaged in activation of gingipain zymogens on the bacterial cell surface.

Comprehensive characterization of an aspen (<i>Populus tremuloides</i>) leaf litter sample that maintained ice nucleation activity for 48 years

Abstract. Decaying vegetation was determined to be a potentially important source of atmospheric ice nucleation particles (INPs) in the early 1970s. The bacterium Pseudomonas syringae was the first microorganism with ice nucleation activity (INA) isolated from decaying leaf litter in 1974. However, the ice nucleation characteristics of P. syringae are not compatible with the characteristics of leaf litter-derived INPs since the latter were found to be sub-micron in size, while INA of P. syringae depends on much larger intact bacterial cells. Here we determined the cumulative ice nucleation spectrum and microbial community composition of the historic leaf litter sample 70-S-14 collected in 1970 that conserved INA for 48 years. The majority of the leaf litter-derived INPs were confirmed to be sub-micron in size and to be sensitive to boiling. Culture-independent microbial community analysis only identified Pseudomonas as potential INA. Culture-dependent analysis identified one P. syringae isolate, two isolates of the bacterial species Pantoea ananatis, and one fungal isolate of Mortierella alpina as having INA among 1170 bacterial colonies and 277 fungal isolates, respectively. Both Pa. ananatis and M. alpina are organisms that produce heat-sensitive sub-micron INPs. They are thus both likely sources of the INPs present in sample 70-S-14 and may represent important terrestrial sources of atmospheric INPs, a conclusion that is in line with other recent results obtained in regard to INPs from soil, precipitation, and the atmosphere.

Fully integrated and slidable paper-embedded plastic microdevice for point-of-care testing of multiple foodborne pathogens

This study presents a slidable paper-embedded plastic microdevice fully integrated with DNA extraction, loop-mediated isothermal amplification (LAMP), and colorimetric detection functionalities. The developed microdevice consists of three layers that allow a sliding movement to mix the sample and reagents for DNA purification, amplification, and detection in a sequential manner. An FTA card was employed in the main chamber for DNA extraction and purification from intact bacterial cells. Subsequently, LAMP reagents and fuchsin-stored chambers were pulled toward the main chambers for DNA amplifications at 65 °C. After 30 min, the detection reagents-stored chambers were then moved to main chambers for result analysis. For the detection of LAMP amplicons, a novel colorimetric fuchsin-based method was employed. The wide applicability of the integrated microdevice was demonstrated by successfully screening three major foodborne pathogens, namely Salmonella spp., Staphylococcus aureus, and Escherichia coli O157:H7 in food, enabling highly sensitive detection of 3.0 × 101 CFU/sample of Gram-negative bacteria (Salmonella spp. and Escherichia coli O157:H7) and 3.0 × 102 CFU/sample of Gram-positive bacteria (Staphylococcus aureus) within 75 min. The portable and integrated microdevice presented in this study holds significant promise for point-of-care applications to accurately and rapidly diagnose and control diseases.

Incomplete cell disruption of resistant microbes

Biomolecules for OMIC analysis of microbial communities are commonly extracted by bead-beating or ultra-sonication, but both showed varying yields. In addition to that, different disruption pressures are necessary to lyse bacteria and fungi. However, the disruption efficiency and yields comparing bead-beating and ultra-sonication of different biological material have not yet been demonstrated. Here, we show that ultra-sonication in a bath transfers three times more energy than bead-beating over 10 min. TEM imaging revealed intact gram-positive bacterial and fungal cells whereas the gram-negative bacterial cells were destroyed beyond recognition after 10 min of ultra-sonication. DNA extraction using 10 min of bead-beating revealed higher yields for fungi but the extraction efficiency was at least three-fold lower considering its larger genome. By our critical viewpoint, we encourage the review of the commonly used extraction techniques as we provide evidence for a potential underrepresentation of resistant microbes, particularly fungi, in ecological studies.

Sand and silty-sand soil stabilization using bacterial enzyme–induced calcite precipitation (BEICP)

This paper examines the bio-derived stabilization of sand-only or sand-plus-silt soils using an extracted bacterial enzyme application to achieve induced calcite precipitation (ICP). As compared to conventional microbial induced calcite precipitation (MICP) methods, which use intact bacterial cells, this strategy that uses free urease catalysts to secure bacterial enzyme–induced calcite precipitation (BEICP) appears to offer an improved means of bio-stabilizing silty-sand soils as compared to that of MICP processing. Several benefits may possibly be achieved with this BEICP approach, including bio-safety, environmental, and geotechnical improvements. Notably, the BEICP bio-stabilization results presented in this paper demonstrate (i) higher rates of catalytic urease activity, (ii) a wider range of application with sand-plus-silt soil applications bearing low-plasticity properties, and (iii) the ability to retain higher levels of soil permeability after BEICP processing. Comparative BEICP versus MICP results for sand-only systems are presented, along with BEICP-based results for stabilized soil mixtures at 90:10 and 80:20 percentile sand:silt ratios. This BEICP method’s ability to obtain unconfined compressive strength results in excess of 1000 kPa with sand-plus-silt soil mixtures is particularly noteworthy.

The impact of species, respiration type, growth phase and genetic inventory on absolute metal content of intact bacterial cells.

Metal ions are abundant in microbial proteins and have structural, catalytic or electron-transferring roles. Metalloproteins are especially prevalent in respiratory chains where they couple electron flow with proton translocation across the membrane. Here, we explore the hypothesis that anaerobic respiratory chains can be investigated by quantitative whole-cell metallomics of the key metals Fe, Co, Ni and Mo. Sensitive and strictly quantitative data were obtained by inductively-coupled plasma mass spectrometry when using a triple quadrupole instrument (ICP-QqQ-MS). Our experiments provide data on the absolute cellular metal content of E. coli, an enrichment culture of "Ca. Kuenenia stuttgartiensis", Dehalococcoides mccartyi, Desulfovibrio vulgaris, Geobacter sulfurreducens and Geobacter metallireducens. A major obstacle in whole-cell metallomics is the interference caused by metal precipitates, observed for G. metallireducens and D. vulgaris. In the other investigated organisms, whole-cell metallomics gave biologically meaningful information, e.g. high Fe and Co content in "Ca. K. stuttgartiensis" and higher Mo content in E. coli when grown under nitrate-reducing conditions. The content of all four metals was almost constant in E. coli from the late exponential phase allowing precise measurements independent of the exact duration of cultivation. Deletion or overexpression of genes involved in metal homeostasis (Ni transport or Mo-cofactor metabolism) was mirrored by dramatic changes in whole-cell metal content. Deletion of genes encoding abundant metalloproteins or heterologous overexpression of metalloproteins was also reflected in the whole-cell metal content. Our study provides a reference point for absolute microbial metallomics and paves the way for the development of fast and easy mutation screens.

Characterization of the bacterial microbiome in first-pass meconium using propidium monoazide (PMA) to exclude nonviable bacterial DNA.

Numerous studies have reported bacterial DNA in first-pass meconium samples, suggesting that the human gut microbiome is seeded prior to birth. However, these studies have not been able to discriminate between DNA from living bacterial cells, DNA from dead bacterial cells or cell-free DNA. Here we have used propidium monoazide (PMA) together with 16S rRNA gene sequencing to determine whether there are intact bacterial cells in the fetal gut. DNA was extracted from first-pass meconium (n=5) and subjected to 16S rRNA gene sequencing with/without PMA treatment. All meconium samples, regardless of PMA treatment, contained detectable levels of bacterial DNA; however, treatment with PMA prior to DNA extraction decreased the DNA yield by approximately 20%. PMA-treated meconium samples did not differ significantly from untreated samples in terms of observed number of OTUs (P=0·945); although they did differ taxonomically, with around one quarter of OTUs identified in untreated samples only, suggesting that they have originated from cell-free/nonviable DNA. The mean Sørensen coefficient for treated vs untreated samples was 0·527. Our findings suggest that the fetal gut is seeded with intact bacterial cells prior to birth. This is an important finding, as exposure to live bacteria during gestation might have a significant impact on the developing fetus. SIGNIFICANCE AND IMPACT OF THE STUDY: DNA-based microbiome studies performed using 16S rRNA gene sequencing are limited by their inability to discriminate between live bacterial cells, dead bacterial cells and cell-free DNA. Here we use propidium monoazide (PMA) to exclude nonviable bacteria from microbiome analysis of first-pass meconium samples and thereby reveal that the majority of the purported fetal gut microbiome is from intact bacterial cells. This work demonstrates the importance of excluding nonviable bacteria when analysing the microbial community in low-biomass samples such as meconium.

Direct Transfer of a Mycoplasma mycoides Genome to Yeast Is Enhanced by Removal of the Mycoides Glycerol Uptake Factor Gene glpF.

We previously discovered that intact bacterial chromosomes can be directly transferred to a yeast host cell where they can propagate as centromeric plasmids by fusing bacterial cells with S accharomyces cerevisiae spheroplasts. Inside the host any desired number of genetic changes can be introduced into the yeast centromeric plasmid to produce designer genomes that can be brought to life using a genome transplantation protocol. Earlier research demonstrated that the removal of restriction-systems from donor bacteria, such as Mycoplasma mycoides, Mycoplasma capricolum, or Haemophilus influenzae increased successful genome transfers. These findings suggested that other genetic factors might also impact the bacteria-to-yeast genome transfer process. In this study, we demonstrated that the removal of a particular genetic factor, the glycerol uptake facilitator protein gene glpF from M. mycoides, significantly increased direct genome transfer by up to 21-fold. Additionally, we showed that intact bacterial cells were endocytosed by yeast spheroplasts producing organelle-like structures within these yeast cells. These might lead to the possibility of creating novel synthetic organelles.

Novel Glycopolymer Eradicates Antibiotic- and CCCP-Induced Persister Cells in Pseudomonas aeruginosa.

Antibiotic treatments often fail to completely eradicate a bacterial infection, leaving behind an antibiotic-tolerant subpopulation of intact bacterial cells called persisters. Persisters are considered a major cause for treatment failure and are thought to greatly contribute to the recalcitrance of chronic infections. Pseudomonas aeruginosa infections are commonly associated with elevated levels of drug-tolerant persister cells, posing a serious threat to human health. This study represents the first time a novel large molecule polycationic glycopolymer, poly (acetyl, arginyl) glucosamine (PAAG), has been evaluated against antibiotic and carbonyl cyanide m-chlorophenylhydrazone induced P. aeruginosa persisters. PAAG eliminated eliminated persisters at concentrations that show no significant cytotoxicity on human lung epithelial cells. PAAG demonstrated rapid bactericidal activity against both forms of induced P. aeruginosa persister cells resulting in complete eradication of the in vitro persister cells within 24 h of treatment. PAAG demonstrated greater efficacy against persisters in vitro than antibiotics currently being used to treat persistent chronic infections such as tobramycin, colistin, azithromycin, aztreonam, and clarithromycin. PAAG caused rapid permeabilization of the cell membrane and caused significant membrane depolarization in persister cells. PAAG efficacy against these bacterial subpopulations suggests it may have substantial therapeutic potential for eliminating recurrent P. aeruginosa infections.

Effects of a small, volatile bacterial molecule on Pseudomonas aeruginosa bacteria using whole cell high-resolution magic angle spinning nuclear magnetic resonance spectroscopy and genomics.

In the present study, high-resolution magic-angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy was applied to live Pseudomonas aeruginosa (PA) bacterial cells to determine the metabolome of this opportunistic Gram-negative human pathogen, and in particular, its response to the volatile aromatic low molecular weight signaling molecule, 2-aminoacetophenone (2-AA). Multi-dimensional HRMAS NMR is a promising method which may be used to determine the in vivo metabolome of live intact bacterial cells; 2-AA is produced by PA and triggers the emergence of phenotypes that promote chronic infection phenotypes in in vitro and in vivo (animal) models. In the present study, we applied one-dimensional and two-dimensional proton (1H) HRMAS NMR to PA cells which were grown with or without 2-AA in order to examine the associations between metabolites and cellular processes in response to 2-AA. We also compared whole-genome transcriptome profiles of PA cells grown with or without 2-AA and found that 2-AA promoted profound metabolic changes in the PA cells. By comparing the whole-genome transcriptome profiles and metabolomic analysis, we demonstrated that 2-AA profoundly reprogramed the gene expression and metabolic profiles of the cells. Our in vivo 1H HRMAS NMR spectroscopy may prove to be a helpful tool in the validation of gene functions, the study of pathogenic mechanisms, the classification of microbial strains into functional/clinical groups and the testing of anti-bacterial agents.