Target Bacterial Cells Research Articles

K+ Ions Crosslinked Kappa-Carrageenan/Methylene Blue Composite Film. Part-1: Synthesis, Characterization and Application as Controlled Release Device for Photodynamic Therapy Application

K+ ions crosslinked (KC/MB) Methylene blue photosensitizer loaded composite film is prepared for photodynamic therapy (PDT) application as Photosensitizer released by composite film can conveniently be controlled by using different quantities of K+ ions and this application is based on dynamic interaction between light with suitable wavelength, photosensitizer and molecular oxygen, promoting the death of the target tissue or bacterial cells. In this work, the Methylene Blue (MB) loaded Carrageenan polymeric film was prepared by the method of direct addition of MB into pre-polymerization solution of Carrageenan (KC). This composite film and plain KC film were characterized by FTIR, XRD and SEM analysis. The characteristic peaks of MB photosensitizer were obtained in FTIR spectrum of composite film. The composite film KC/MB shows prominence of amorphous nature may be due to MB is present in very small quantity. The SEM analysis reveals that the composite film (KC/MB) having much more smooth surface texture throughout the film. Finally, the result of effect of variation in degree of crosslinking on amount of MB release is, as the amount of K+ ions used to crosslink the film increases, MB release decreases.

Development of a chemical scaffold for inhibiting nonribosomal peptide synthetases in live bacterial cells.

The adenylation (A) domain is essential for non-ribosomal peptide synthetases (NRPSs), which synthesize various peptide-based natural products, including virulence factors, such as siderophores and genotoxins. Hence, the inhibition of A-domains could attenuate the virulence of pathogens. 5'-O-N-(Aminoacyl or arylacyl)sulfamoyladenosine (AA-AMS) is a bisubstrate small-molecule inhibitor of the A-domains of NRPSs. However, the bacterial cell permeability of AA-AMS is typically a problem owing to its high hydrophilicity. In this study, we investigated the influence of a modification of 2'-OH in the AMS scaffold with different functional groups on binding to target enzymes and bacterial cell penetration. The inhibitor 7 with a cyanomethyl group at 2'-OH showed desirable inhibitory activity against both recombinant and intracellular gramicidin S synthetase A (GrsA) in the gramicidin S-producer Aneurinibacillus migulanus ATCC 9999, providing an alternative scaffold to develop novel A-domain inhibitors.

A new Rogue-like Escherichia phage UDF157lw to control Escherichia coli O157:H7.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is one of the notorious foodborne pathogens causing high mortality through the consumption of contaminated food items. The food safety risk from STEC pathogens could escalate when a group of bacterial cells aggregates to form a biofilm. Bacterial biofilm can diminish the effects of various antimicrobial interventions and enhance the pathogenicity of the pathogens. Therefore, there is an urgent need to have effective control measurements. Bacteriophages can kill the target bacterial cells through lytic infection, and some enzymes produced during the infection have the capability to penetrate the biofilm for mitigation compared to traditional interventions. This study aimed to characterize a new Escherichia phage vB_EcoS-UDF157lw (or UDF157lw) and determine its antimicrobial efficacy against E. coli O157:H7. Phage characterization included biological approaches, including phage morphology, one-step growth curve, stability tests (pH and temperature), and genomic approaches (whole-genome sequencing). Later, antimicrobial activity tests, including productive infection against susceptible bacterial strains, in vitro antimicrobial activity, and anti-biofilm, were conducted. UDF157lw is a new member of the phages belonging to the Rogunavirus genus, comprising a long and non-contractile tail, isolated from bovine feces and shares close genomic evolutionary similarities with Escherichia phages vB_EcoS-BECP10 and bV_EcoS_AKS96. When used against E. coli O157:H7 (ATCC35150), phage UDF157lw exhibited a latent period of 14 min and a burst size of 110 PFU per infected cell. The phage remained viable in a wide range of pH values (pH 4-11) and temperatures (4-60°C). No virulence genes, such as stx, lysogenic genes, and antibiotic resistance genes, were found. Phage UDF157lw demonstrated high infection efficiencies against different E. coli O157:H7 and generic E. coli strains. In addition, UDF157lw encoded a unique major tail protein (ORF_26) with prominent depolymerase enzyme activity against various E. coli O157:H7 strains, causing large plaque sizes. In contrast to the phage without encoding depolymerase gene, UDF157lw was able to reduce the 24-h and 48-h E. coli O157:H7 biofilm after 1-h phage treatment. The findings of this study provide insights into a new member of the Rogunavirus phages and demonstrate its antimicrobial potential against E. coli O157:H7 in vitro.

Highly specific and sensitive detection of bacteria by dark-field light-scattering imaging based on bacteriophage-modified magnetoplasmonic nanoparticles

Abstract Bacterial infections pose significant global health concerns, necessitating precise and sensitive detection methods. This study introduces a multifunctional probe for bacterial detection. Bifunctional magnetoplasmonic nanoparticles (NPs) serve a dual purpose as carriers for magnetic separation (MS) and enhancers for light scattering of the target bacteria. We achieved exceptional selectivity at the bacterial species level by bioconjugating them with bacteriophages (phages). Optimal phage coverage was carefully determined to enhance the capture efficiency of the target bacterial cells. Additionally, we identified the ideal mixing ratios of the phage probe to bacteria, ensuring efficient MS and high light-scattering intensity. The use of the phage probe enabled the successful separation of captured target bacteria from other bacterial strains in the sample mixture. Bacterial detection was accomplished through dark-field light-scattering imaging, eliminating the need for additional labeling with other probes. This method showed a remarkable sensitivity, achieving a detection limit of ∼102 colony-forming units ml−1. This value is 2 orders of magnitude lower than that achieved using our previously reported technique using monofunctional plasmonic NPs. These phage probes hold promise for healthcare, biotech, and environmental monitoring owing to their adaptability to diverse bacterial species.

The Application of Bacteriophage and Photoacoustic Flow Cytometry in Bacterial Identification.

Infection with resistant bacteria has become an ever-increasing problem in modern medical practice. Bacteremia is a serious and potentially lethal condition that can lead to sepsis without early intervention. Currently, broad-spectrum antibiotics are prescribed until bacteria can be identified through blood cultures, a process that can take 2-3 days and is unable to provide quantitative information. Staphylococcus aureus (S. aureus) is a leading cause of bacteremia, and methicillin-resistant S. aureus (MRSA) accounts for more than a third of the cases. Other bacteria such as Clostridium difficile, Acinetobacter baumannii, and Carbapenem-resistant Enterobacteriaceae are becoming more prevalent and antibiotic-resistant. Rapid diagnostics for each of these superbugs has been a priority for health organizations around the world. Bacteriophages have evolved for millions of years to develop exquisite specificity in target binding using their host attachment proteins. Bacteriophages are viruses that infect bacteria. Bacteriophages use tail spikes, specialized attachment proteins, to bind specifically to their target bacterial cell surface proteins. We use bacteriophages and parts of bacteriophages as specific tags coupled with photoacoustic flow cytometry for the detection and quantification of bacteria. In photoacoustic flow cytometry, laser light is absorbed by particles under flow, and the ultrasound waves generated on the release of the energy are detected. Photoacoustics involves the detection of ultrasound waves resulting from laser irradiation. In photoacoustic flow cytometry, pulsed laser light is delivered to a sample flowing past a focused transducer, and particles that absorb laser light create a photoacoustic response. Bacteria can be tagged with dyed bacteriophage and processed through a photoacoustic flow cytometer where they are detected by the acoustic response. In this chapter, we describe the procedure and methods used to accomplish this. Often the limiting factor for the treatment of patients is the time spent waiting for results. It is our hope that the work presented in this chapter can be a foundation for future work and provide an ability to detect bacterial pathogens in blood cultures. Bacterial plate cultures and Gram staining are nineteenth-century technologies that have been the gold standards for decades, but current trends in resistant bacteria have necessitated a move toward more rapid and quantifiable diagnostic tools.

Single-Cell Detection of Erwinia amylovora Using Bio-Functionalized SIS Sensor.

Herein, we developed a bio-functionalized solution-immersed silicon (SIS) sensor at the single-cell level to identify Erwinia amylovora (E. amylovora), a highly infectious bacterial pathogen responsible for fire blight, which is notorious for its rapid spread and destructive impact on apple and pear orchards. This method allows for ultra-sensitive measurements without pre-amplification or labeling compared to conventional methods. To detect a single cell of E. amylovora, we used Lipopolysaccharide Transporter E (LptE), which is involved in the assembly of lipopolysaccharide (LPS) at the surface of the outer membrane of E. amylovora, as a capture agent. We confirmed that LptE interacts with E. amylovora via LPS through in-house ELISA analysis, then used it to construct the sensor chip by immobilizing the capture molecule on the sensor surface modified with 3'-Aminopropyl triethoxysilane (APTES) and glutaraldehyde (GA). The LptE-based SIS sensor exhibited the sensitive and specific detection of the target bacterial cell in real time. The dose-response curve shows a linearity (R2 > 0.992) with wide dynamic ranges from 1 to 107 cells/mL for the target bacterial pathogen. The sensor showed the value change (dΨ) of approximately 0.008° for growing overlayer thickness induced from a single-cell E. amylovora, while no change in the control bacterial cell (Bacillus subtilis) was observed, or negligible change, if any. Furthermore, the bacterial sensor demonstrated a potential for the continuous detection of E. amylovora through simple surface regeneration, enabling its reusability. Taken together, our system has the potential to be applied in fields where early symptoms are not observed and where single-cell or ultra-sensitive detection is required, such as plant bacterial pathogen detection, foodborne pathogen monitoring and analysis, and pathogenic microbial diagnosis.

Identification and distribution of new candidate T6SS effectors encoded in Salmonella Pathogenicity Island 6.

The type VI secretion system (T6SS) is a contact-dependent contractile multiprotein apparatus widely distributed in Gram-negative bacteria. These systems can deliver different effector proteins into target bacterial and/or eukaryotic cells, contributing to the environmental fitness and virulence of many bacterial pathogens. Salmonella harbors five different T6SSs encoded in different genomic islands. The T6SS encoded in Salmonella Pathogenicity Island 6 (SPI-6) contributes to Salmonella competition with the host microbiota and its interaction with infected host cells. Despite its relevance, information regarding the total number of effector proteins encoded within SPI-6 and its distribution among different Salmonella enterica serotypes is limited. In this work, we performed bioinformatic and comparative genomics analyses of the SPI-6 T6SS gene cluster to expand our knowledge regarding the T6SS effector repertoire and the global distribution of these effectors in Salmonella. The analysis of a curated dataset of 60 Salmonella enterica genomes from the Secret6 database revealed the presence of 23 new putative T6SS effector/immunity protein (E/I) modules. These effectors were concentrated in the variable regions 1 to 3 (VR1-3) of the SPI-6 T6SS gene cluster. VR1-2 were enriched in candidate effectors with predicted peptidoglycan hydrolase activity, while VR3 was enriched in candidate effectors of the Rhs family with C-terminal extensions with predicted DNase, RNase, deaminase, or ADP-ribosyltransferase activity. A global analysis of known and candidate effector proteins in Salmonella enterica genomes from the NCBI database revealed that T6SS effector proteins are differentially distributed among Salmonella serotypes. While some effectors are present in over 200 serotypes, others are found in less than a dozen. A hierarchical clustering analysis identified Salmonella serotypes with distinct profiles of T6SS effectors and candidate effectors, highlighting the diversity of T6SS effector repertoires in Salmonella enterica. The existence of different repertoires of effector proteins suggests that different effector protein combinations may have a differential impact on the environmental fitness and pathogenic potential of these strains.

Evaluation of deoxythymidine-based cationic amphiphiles as antimicrobial, antibiofilm, and anti-inflammatory agents

ObjectivesWe recently designed a series of cationic deoxythymidine-based amphiphiles that mimic the cationic amphipathic structure of antimicrobial peptides (AMPs). Among these amphiphiles, ADG-2e and ADL-3e displayed the highest selectivity against bacterial cells. In this study, ADG-2e and ADL-3e were evaluated for their potential as novel classes of antimicrobial, antibiofilm, and anti-inflammatory agents. MethodsMinimum inhibitory concentrations of ADG-2e and ADL-3e against bacteria were determined using the broth microdilution method. Proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K was determined by radial diffusion and HPLC analysis. Biofilm activity was investigated using the broth microdilution and confocal microscopy. The antimicrobial mechanism was investigated by membrane depolarization, cell membrane integrity analysis, scanning electron microscopy (SEM), genomic DNA influence and genomic DNA binding assay. Synergistic activity was evaluated using checkerboard method. Anti-inflammatory activity was investigated using ELISA and RT-PCR. ResultsADG-2e and ADL-3e showed good resistance to physiological salts and human serum, and a low incidence of drug resistance. Moreover, they exhibit proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K. ADG-2e and ADL-3e were found to kill bacteria by an intracellular target mechanism and bacterial cell membrane-disrupting mechanism, respectively. Furthermore, ADG-2e and ADL-3e showed effective synergistic effects when combined with several conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Importantly, ADG-2e and ADL-3e not only suppressed MDRPA biofilm formation but also effectively eradicated mature MDRPA biofilms. Furthermore, ADG-2e and ADL-3e drastically decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expression and protein secretion in lipopolysaccharide (LPS)-stimulated macrophages, implying potent anti-inflammatory activity in LPS-induced inflammation. ConclusionOur findings suggest that ADG-2e and ADL-3e could be further developed as novel antimicrobial, antibiofilm, and anti-inflammatory agents to combat bacterial infections.

Green algae-derived triple CATH_BRALE multimer protein potently inhibits bacterial growth by permeabilizing the bacterial cell membrane

Salmonoid cathelicidin (termed CATH_BRALE) is a fish-derived antimicrobial peptide (AMP) isolated from Brachymystax lenok. In this study, CATH_BRALE was expressed in Chlamydomonas reinhardtii as a format of three repeats (3×CATH_BRALE) attached with hemagglutinin (HA) and 6×His at its C-terminus, producing a recombinant peptide with a molecular weight of ∼18 kDa. The recombinant 3×CATH_BRALE-HA-6×His remains to be expressed stably in Chlamydomonas cells even after passaging continuously for five months and yields up to 0.21% of the total Chlamydomonas soluble proteins. 3×CATH_BRALE-HA-6×His showed broad-spectrum antibacterial activity against bacteria, with MIC values ranging from 40 to 50 µg/ml for Gram-negative bacteria and 30–40 µg/ml for Gram-positive bacteria. This recombinant peptide had strong thermostability and pH stability, and its antibacterial activity was rarely altered when temperature and pH are changed. It resisted to the digestion of several tested proteases to certain extents. Besides, 3×CATH_BRALE-HA-6×His is biologically safe as it did not hemolyze rat erythrocytes nor caused cytotoxicity on Vero, BHK21, HEK293, and MDBK cells. Its antibacterial action was achieved by penetrating the cell membrane to disrupt the membrane of the target bacterial cell. In sum, our data showed that C. reinhardtii can be used as a heterologous expression host to produce biologically active CATH_BRALE.

Development of Host-Cleavable Antibody-Bactericide Conjugates against Extracellular Pathogens.

Novel antimicrobial agents with potent bactericidal activity are needed to treat infections caused by multidrug-resistant (MDR) extracellular pathogens, such as Pseudomonas aeruginosa. Antimicrobial peptides (AMPs) and peptidomimetics are promising alternatives to traditional antibiotics, but their therapeutic use is limited due to the lack of specificity and resulting off-target effects. The incorporation of an antibody into the drug design would alleviate these challenges by localizing the AMP to the target bacterial cells. Antibody-drug conjugates (ADCs) have already achieved clinical success as anticancer therapeutics, due to the ability of the antibody to deliver the payload directly to the cancer cells. This strategy involves the selective delivery of highly cytotoxic drugs to the target cells, which enables a broad therapeutic window. This platform can be translated to the treatment of infections, whereby an antibody is used to deliver an antimicrobial agent to the bacterial antigen. Herein, we propose the development of an antibody-bactericide conjugate (ABC) in which the antibacterial oligothioetheramide (oligoTEA), BDT-4G, is coupled to an anti-P. aeruginosa antibody via a cleavable linker. The drug BDT-4G was chosen based on its efficacy against a range of P. aeruginosa isolates and its ability to evade mechanisms conferring resistance to the last-resort agent polymyxin B. We demonstrate that the ABC binds to the bacterial cell surface, and following cleavage of the peptide linker, the oligoTEA payload is released and exhibits antipseudomonal activity.

A New Kayfunavirus-like Escherichia Phage vB_EcoP-Ro45lw with Antimicrobial Potential of Shiga Toxin-Producing Escherichia coli O45 Strain.

Lytic bacteriophages are re-considered as a solution to resolve antibiotic-resistant rampage. Despite frequent foodborne outbreaks caused by the top six non-O157 Shiga-toxin-producing Escherichia coli (STEC), the current interventions are not sufficiently effective against each serogroup, particularly O45. Therefore, this study aimed to characterize a new short-tailed phage, vB_EcoP-Ro45lw (or Ro45lw), as an alternative antimicrobial agent for STEC O45 strains. Phage Ro45lw belongs to the Kayfunavirus genus within the Autographiviridae family and shares no close evolutionary relationship with any reference phages. Ro45lw contains a tail structure composed of a unique tail fiber and tail tubular proteins A and B, likely to produce enzymatic activity against the target bacterial cells besides structural function. Additionally, the phage genome does not contain virulent, antibiotic-resistant, or lysogenic genes. The phage has a latent period of 15 min with an estimated burst size of 55 PFU/CFU and is stable at a wide range of pH (pH4 to pH11) and temperatures (30 °C to 60 °C). Regardless of the MOIs (MOI = 0.1, 1, and 10) used, Ro45lw has a strong antimicrobial activity against both environmental (E. coli O45:H-) and clinical (E. coli O45:H2) strains at 25 °C. These findings indicate that phage Ro45lw has antimicrobial potential in mitigating pathogenic STEC O45 strains.

Plant-derived nanoparticles as alternative therapy against Diarrheal pathogens in the era of antimicrobial resistance: A review.

Diarrhea is a condition in which feces is discharged from the bowels frequently and in a liquid form. It is one of the frequent causes of morbidity and mortality in developing countries. The impact of Diarrhea is worsened by the increasing incidence of antimicrobial resistance among the causative agents, and this is now categorized as a global healthcare challenge. Antimicrobial resistance among Diarrheal pathogens also contributes to extended infection durations, and huge economic loss even in countries with advanced public health policies. The ever-increasing incidence of antimicrobial resistance including the contraindications arising from the administration of antibiotics in some Diarrheal cases highlights a crucial need for the development of novel non-antibiotic alternative agents for therapeutic and biocontrol applications. One such intervention includes the application of plant-derived nanoparticles (PDNPs) with novel antimicrobial properties. Given their small size and large surface area to volume ratio, PDNPs can attack target bacterial cell walls to generate reactive oxygen species that may simultaneously disrupt bacteria cell components such as DNA and proteins leading to cell damage or death. This potential can make it very difficult for pathogenic organisms to develop resistance against these antibacterial agents. In this review, we provide a critical overview on the antimicrobial resistance crisis among Diarrheagenic bacteria. We also discuss the evidence from the existing literature to support the potential associated with the use of PDNPs as alternative therapeutic agents for multidrug resistant and antibiotics administer contraindicated bacteria that are associated with Diarrhea.

Suppression of the insect cuticular microbiomes by a fungal defensin to facilitate parasite infection.

Insects can assemble defensive microbiomes on their body surfaces to defend against fungal parasitic infections. The strategies employed by fungal pathogens to combat host cuticular microbiotas remains unclear. Here, we report the identification and functional characterization of the defensin-like antimicrobial gene BbAMP1 encoded by the entomopathogenic fungus Beauveria bassiana. The mature peptide of BbAMP1 can coat fungal spores and can be secreted by the fungus to target and damage Gram-positive bacterial cells. Significant differences in insect survival were observed between the wild-type and BbAMP1 mutant strains during topical infection but not during injection assays that bypassed insect cuticles. Thus, BbAMP1 deletion considerably reduced fungal virulence while gene overexpression accelerated the fungal colonization of insects compared with the wild-type strain in natural infections. Topical infection of axenic Drosophila adults evidenced no difference in fly survivals between strains. However, the gnotobiotic infections with the addition of Gram-positive but not Gram-negative bacterial cells in fungal spore suspensions substantially increased the survival of the flies treated with ∆BbAMP1 compared to those infected by the wild-type and gene-overexpression strains. Bacterial colony counts and microbiome analysis confirmed that BbAMP1 could assist the fungus to manipulate insect surface bacterial loads. This study reveals that fungal defensin can suppress the host surface defensive microbiomes, which underscores the importance to extend the research scope of fungus-host interactions.

Antimicrobial and immunoregulatory activities of the derived peptide of a natural killer lysin from black rockfish (Sebastes schlegelii)

Natural killer lysin (NK-lysin) is a small molecule antimicrobial peptide secreted by natural killer cells and T lymphocytes. In this study, we characterized a cDNA sequence encoding an NK-lysin homologue (SsNKL1) from black rockfish, Sebastes schlegelii. The open reading frame (ORF) of SsNKL1 encodes a putative protein of 149 amino acids and shares 44%–87% overall sequence identities with other teleost NK-lysins. SsNKL1 possesses conserved NK-lysin family features, including a signal sequence and a surfactant-associated protein B (SapB) domain, sequence analysis revealed that SsNKL1 is most closely related to false kelpfish (Sebastiscus marmoratus) NK-lysin (with 87% sequence identity). SsNKL1 transcripts were detected in all the tested tissues, with the highest level in the kidney, followed by the spleen and gills. Upon Listonella anguillarum infection, the mRNA expression of SsNKL1 in the black rockfish was significantly up-regulated in the liver and kidney. The derived peptide SsNKLP27 from SsNKL1 was synthesized, and its biological function was studied. SsNKLP27 showed direct antibacterial activity against Gram-negative and Gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, L. anguillarum, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio vulnificus. SsNKLP27 treatment facilitated the bactericidal process of erythromycin by enhancing the permeability of the outer membrane. In the process of interaction with the target bacterial cells, SsNKLP27 changed the permeability and retained the morphological integrity of the cell membrane, then penetrated into the cytoplasm, and induced the degradation of genomic DNA and total RNA. In vivo studies showed that administration of SsNKLP27 before bacterial and viral infection significantly reduced the transmission and replication of pathogens in tissues. In vitro analysis showed that SsNKLP27 could enhance the respiratory burst ability and regulate the expression of some immune-related genes of macrophages. In summary, these results provided new insights into the function of NK-lysins in teleost fish and support that SsNKLP27 is a new broad-spectrum antimicrobial peptide that has a potential application prospect in aquaculture against pathogenic infection.

Evaluation of Bacteriophage-Antibiotic Combination Therapy for Biofilm-Embedded MDR Enterococcus faecium.

Multidrug-resistant (MDR) Enterococcus faecium is a challenging pathogen known to cause biofilm-mediated infections with limited effective therapeutic options. Lytic bacteriophages target, infect, and lyse specific bacterial cells and have anti-biofilm activity, making them a possible treatment option. Here, we examine two biofilm-producing clinical E. faecium strains, daptomycin (DAP)-resistant R497 and DAP-susceptible dose-dependent (SDD) HOU503, with initial susceptibility to E. faecium bacteriophage 113 (ATCC 19950-B1). An initial synergy screening was performed with modified checkerboard MIC assays developed by our laboratory to efficiently screen for antibiotic and phage synergy, including at very low phage multiplicity of infection (MOI). The data were compared by one-way ANOVA and Tukey (HSD) tests. In 24 h time kill analyses (TKA), combinations with phage-DAP-ampicillin (AMP), phage-DAP-ceftaroline (CPT), and phage-DAP-ertapenem (ERT) were synergistic and bactericidal compared to any single agent (ANOVA range of mean differences 3.34 to 3.84 log10 CFU/mL; p < 0.001). Furthermore, phage-DAP-AMP and phage-DAP-CPT prevented the emergence of DAP and phage resistance. With HOU503, the combination of phage-DAP-AMP showed the best killing effect, followed closely by phage-DAP-CPT; both showed bactericidal and synergistic effects compared to any single agent (ANOVA range of mean differences 3.99 to 4.08 log10 CFU/mL; p < 0.001).

Development of highly sensitive optical nanoantenna for bacterial detection.

Gold nanoparticles (AuNPs) are chemically stable and serve as excellent labels because their characteristic red coloration based on the localized surface plasmon resonance (LSPR) does not fade. However, it is necessary to control the structure of AuNPs to use them as labels for various analyses, because their optical properties depend strongly on their size, shape, and state of aggregation. In this study, we developed gold nanostructures (AuNSs) by encapsulating many small AuNPs within a polymer for scattering light-based bacterial detection. The AuNSs consisting of many small nanoparticles provided stronger scattered light intensity than a single AuNP of the same particle size. We found that the aggregation of the AuNSs enhanced the scattering light intensity, depending strongly on their aggregation states, and did not affect the wavelength of the scattering light observed under a dark-field microscope. By specifically binding the antibody-introduced AuNSs to the antigen on the bacterial surface, it was possible to label the target bacteria and detect them based on their light scattering characteristics. In addition, to improve the accuracy of the selective identification of the cells of interest, labels based on scattered light should ideally have a fixed wavelength of scattered light with high intensity. From these perspectives, we developed a method of constructing an optical antenna on the surface of target bacterial cells using antibody-introduced NSs.

More Than Just a Spearhead: Diverse Functions of PAAR for Assembly and Delivery of Toxins of the Contractile Injection Systems.

ABSTRACTThe type VI secretion system (T6SS) belongs to the evolutionarily related group of contractile injection systems that employ a contractile outer sheath to inject a rigid spear-like inner tube into target bacterial and eukaryotic cells. The tip of the rigid tube is often decorated by a PAAR-repeat protein as a key structural component. Many members of the PAAR protein family can also have additional and diverse functions by serving as toxins for those with extended domains or as carriers for interacting toxins. A plethora of toxin modules or modules of unknown functions have been bioinformatically predicted to be associated with PAAR either as a fused domain or as an interacting partner, and yet only a small number of PAAR proteins have been studied, highlighting the exciting and dire need for future research to better understand the diverse PAAR-mediated functions.

Time-kill kinetics and mechanism of action of Caesalpinia sappan L. and Ochna integerrima (Lour.) Merr. water extracts against pathogenic bacteria

Context: Caesalpinia sappan and Ochna integerrima are two native plants of Thailand and distributed widely throughout the country. Recently, water extracts from the stem bark of C. sappan and O. integerrima were found to exhibit potently antibacterial property but the antibacterial mechanism of action toward target bacteria has not been investigated yet. Aims: To study time-kill kinetic and the mechanism of action of C. sappan and O. integerrima waterextracts toward target bacteria. Methods: The time-kill kinetic study of bacteriostatic or bactericidal activity against Gram-positive and Gram-negative pathogenic bacteria within 24 h experiment was conducted and the mechanism of action on cell morphology of the target bacteria by using scanning electron microscopy (SEM) were investigated. Results: Both of the C. sappan and O. integerrima water extracts possessed bacteriostatic or bactericidal activity toward six tested pathogenic bacteria. The bacteriostatic or bactericidal activity depending on bacterial strains and the test concentrations (0.5, 1 and 2 × MIC) of these two extracts to the tested bacteria were observed. The bactericidal activity against all of the tested bacterial was evinced at 2 × MIC. The morphological alterations of the target bacterial cells of changing the cell size (decreased and increased), cell lysis and having cell cavity were detected by SEM after the treatment with C. sappan and O. integerrima water extracts. Conclusions: The water extracts from the stem bark of C. sappan and O. integerrima exhibited bactericidal activity by changing the cell size, cell lysis and cell cavity of the tested bacteria cell morphology.

Listeriolysin S: A bacteriocin from Listeria monocytogenes that induces membrane permeabilization in a contact-dependent manner

Listeriolysin S (LLS) is a thiazole/oxazole-modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism.

Catalytic ferromagnetic gold nanoparticle immunoassay for the detection and differentiation of Mycobacterium tuberculosis and Mycobacterium bovis

A ferromagnetic gold nanoparticle based immune detection assay, exploiting the enhanced signal amplification of inorganic nanozymes, was developed and evaluated for its potential application in the detection of Mycobacterium tuberculosis complex (MTBC) organisms, and simultaneous identification of Mycobacterium bovis. Ferromagnetic gold nanoparticles (Au–Fe3O4 NPs) were prepared and their intrinsic peroxidase-like activity exploited to catalyse 3,3′,5’,5-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). When the Au–Fe3O4 NPs were functionalised by direct coupling with MTBC-selective antibodies, a nanoparticle based immune detection assay (NPIDA) was developed which could detect Mycobacterium tuberculosis (MTB) and differentiate M. bovis. In the assay, the intrinsic magnetic capability of the functionalised Au–Fe3O4 NPs was used in sample preparation to capture target bacterial cells. These were incorporated into a novel immunoassay which used species selective monoclonal antibodies (mAb) to detect bound target. The formation of a blue TMB oxidation product, with a peak absorbance of 370 nm, indicated successful capture and identification of the target. The detection limit of the NPIDA for both MTB and M. bovis was determined to be comparable to conventional ELISA using the same antibodies. Although limited matrix effects were observed in either assay, the NPIDA offers a reduced time to confirmatory identification.This novel NPIDA was capable of simultaneous sample concentration, purification, immunological detection and speciation. To our knowledge, it represents the first immune-based diagnostic test capable of identifying MTBC organisms and simultaneously differentiating M. bovis.