Studying the viability and growth kinetics of vancomycin-resistant Enterococcus faecalis V583 following femtosecond laser irradiation (420-465 nm).

Enterococcus faecalis is among the most resistant bacteria found in infected root canals. The demand for cutting-edge disinfection methods has rekindled research on photoinactivation with visible light. This study investigated the bactericidal activity of femtosecond laser irradiation against vancomycin-resistant Enterococcus faecalis V583 (VRE). The effect of parameters such as wavelength and energy density on the viability and growth kinetics of VRE was studied to design an optimized laser-based antimicrobial photoinactivation approach without any prior addition of exogenous photosensitizers. The most effective wavelengths were 430 nm and 435 nm at a fluence of 1000 J/cm2, causing a nearly 2-log reduction (98.6% and 98.3% inhibition, respectively) in viable bacterial counts. The colony-forming units and growth rate of the laser-treated cultures were progressively decreased as energy density or light dose increased at 445 nm but reached a limit at 1250 J/cm2. At a higher fluence of 2000 J/cm2, the efficacy was reduced due to a photobleaching phenomenon. Our results highlight the importance of optimizing laser exposure parameters, such as wavelength and fluence, in bacterial photoinactivation experiments. To our knowledge, this is the first study to report an optimized wavelength for the inactivation of VRE using visible femtosecond laser light.

Phosphorylation-amplified synchronized droplet microfluidics sensitizes bacterial growth kinetic real-time monitoring.

The necessity for rapid and accurate bacterial growth monitoring is imperative across various domains, including healthcare and environmental safety. We introduce the self-synchronized droplet-amplified electrical screening cytometry (SYNC) system, a novel meld of droplet microfluidics and electrochemical amplification tailored for precise bacterial growth kinetic monitoring. SYNC encapsulates single bacteria in picolitre droplets, enabling real-time, fluorescence-free electrochemical monitoring. A specially devised phosphorylation-amplified culture medium translates bacterial metabolic activity into discernible electrical impedance changes. The dual-channel design and a rail-based structure in SYNC facilitate parallel screening and self-synchronization of droplets, addressing the limitations of conventional impedance cytometry. SYNC showcases a 5-fold enhancement in detection sensitivity and reduces 50% of the detection time compared to traditional approaches. Notably, SYNC is pioneering in providing exact initial bacterial concentrations, achieve to 104 bacteria/ml, a capability unmatched by existing real-time techniques measuring electrochemical variations. Along with its robust performance, this earmarks SYNC as a powerful tool for applications such as antibiotic susceptibility testing, food quality monitoring, and real-time water bacteria monitoring, paving the way for enhanced microbial process management and infection control.

Cytolysin A-mediated protein exportation efficiency and its role in enhancing the fitness of live recombinant Salmonella Typhi vaccine strain.

The genetic fusion of cytolysin A (clyA) to heterologous antigen expressed in live Salmonella vector demonstrated efficient translocation into periplasmic space and extracellular medium. Accumulating evidence has shown that clyA-mediated antigen delivery improved growth fitness and enhanced immunogenicity of live vector vaccine, but the factors influencing this protein exportation has not been investigated. In this study, Toxoplasma gondii antigen fused at C-terminal of clyA protein was expressed in live S. Typhi vector via both plasmid and chromosomal-based expressions. The bivalent strains showed comparable growth rates as monovalent strains, but in varies antigen exportation efficiency. ClyA-fusion antigen with positive charges was translocated to the extracellular spaces, whereas those with negative charges were retained in the cytoplasm. Furthermore, excessive cellular resources expenditure on antigen expression, especially antigen with larger size, could limit the clyA-fusion antigen exportation, resulting in undesirable metabolic burden that eventually affects the growth fitness. Altogether, the present work indicates potential linkage of factors mainly on antigen properties and expression platforms that may affect clyA-mediated antigen delivery to enhance the growth fitness of live vector strain.

Garlic: An Alternative Treatment for Group B Streptococcus.

Prenatal screening in pregnant women between 35 and 37 weeks of gestation and intrapartum antibiotic prophylaxis has successfully reduced the incidence of neonatal morbidity and mortality related to Streptococcus agalactiae. However, the contamination rates of newborns are still considerable. In traditional and folk medicines, it has been observed that garlic has been effective in treating S. agalactiae infection. The aim of this study was to isolate and identify the active compounds from garlic that have antimicrobial activity against S. agalactiae. In order to do this, SP80 (Sep-Pak 80%) obtained from crude garlic extract (CGE) was fractionated by reverse-phase ultrafast liquid chromatography with UV (RP-UFLC-UV) using a Shim-pack PREP-ODS column. All fractions obtained were tested using a microbial growth inhibition test against the S. agalactiae strain (ATCC 12386). Five clinical isolates were used to confirm the action of the fractions with antimicrobial activity, and the bacterial growth curve was determined. Identification of the antimicrobial compounds was carried out through liquid chromatography coupled with mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR). The active compounds found to exhibit antimicrobial activity were Ƴ-glutamyl-S-allyl-cysteine (fraction 18), Ƴ-glutamyl-phenylalanine (fraction 20), and the two stereoisomers (E and Z) of ajoene (fraction 42). The MICs of these fractions were 5.41 mg/ml, 4.60 mg/ml, and 0.16 mg/ml, respectively, and they inhibited the growth of the clinical isolates tested. Antimicrobial compounds from garlic may be a promising source in the search for new drugs against S. agalactiae. IMPORTANCE Invasive disease due to group B streptococcal (GBS) infection results in a wide spectrum of clinical disease in neonates. Maternal colonization by GBS is the primary risk factor for disease. The strategy recommended by the Centers for Disease Control to reduce neonatal GBS infection is the culture-based screening of all pregnant women at 35 to 37 weeks of gestation and intrapartum antibiotic prophylaxis (IAP). However, indiscriminate use of antibiotics favors the selection and spread of resistant bacteria. The global scenario of antibacterial resistance has been of great concern for public health, and natural products can be a source of new substances to help us grapple with this problem.

The effect of femtosecond laser irradiation on the growth kinetics of Staphylococcus aureus: An in vitro study.

We investigated the effect of femtosecond laser irradiation on the growth kinetics of Staphylococcus aureus. In order to improve laser-based antimicrobial therapy and develop a clinically viable modality, various laser parameters such as laser light wavelength, laser power, exposure time, and energy density were studied. The INSPIRE HF100 laser system (Spectra Physics) provided the femtosecond laser light, which was pumped by a mode-locked femtosecond Ti: sapphire laser MAI TAI HP (Spectra Physics). The survival of the bacterial cells was monitored after irradiation by determination of growth rate using optical density, which is a rapid, simple, and reliable method. The growth rate of laser-exposed cultures was compared to control cultures. Fifteen minutes of exposure to femtosecond laser radiation with a wavelength of 390 nm and 400 nm at an average power of 50 mW was enough to significantly reduce bacterial viability, with a lag in the growth phase of 5 h longer than the control culture (P < 0.0001 by ANOVA and Tukey test).

Virstatin-Conjugated Gold Nanoparticle with Enhanced Antimicrobial Activity against the Vibrio cholerae El Tor Biotype.

Because of the emergence of multidrug-resistant pathogenic bacteria, there is a growing interest for the development of an efficient alternative to antibiotics. Gold nanoparticles (AuNPs) are promising candidates due to their inherent non-toxicity and can be used as effective carriers of drugs. Cholera caused by Gram-negative Vibrio cholerae is still a potential threat in many developing countries. Virstatin, a small molecule, has been reported to inhibit virulence regulation in V. cholerae. Herein, we report an efficient synthesis of virstatin-conjugated gold nanoparticles (VL-AuNPs) and their antibacterial efficacy against the El Tor biotype of V. cholerae (VcN16961). The spherical-shaped NPs have an average diameter of ∼17 nm. The uniqueness of VL-AuNPs relies in the enhanced antibacterial efficacy compared to virstatin, as evidenced from the inhibitory concentration obtained from growth kinetics, and attributed to the inhibition of ATPase activity and DNA damage. More importantly, the expression of cholera toxin, the most important virulence factor of V. cholera, is reduced to a far greater extent than by any of the component molecules. The effect of VL-AuNPs on VcN16961 was monitored using various assays such as confocal microscopy, FACS, fluorescence spectroscopy, and so on. Overall, VL-AuNPs could be a potential candidate for the use as an effective agent for combating diarrheal diseases caused by V. cholera.

Garlic: an alternative treatment for group B Streptococcus

Prenatal screening in pregnant women between 35 and 37 weeks of gestation and intrapartum antibiotic prophylaxis has successfully reduced the incidence of neonatal morbidity and mortality related to Streptococcus agalactiae. However, the contamination rates of newborns are still considerable. In traditional and folk medicines, it has been observed that garlic has been effective in treating S. agalactiae infection. The aim of this study was to isolate and identify the active compounds from garlic that have antimicrobial activity against S. agalactiae. In order to do this, SP80 (Sep-Pak 80%) obtained from crude garlic extract (CGE) was fractionated by reverse-phase ultrafast liquid chromatography with UV (RP-UFLC-UV) using a Shim-pack PREP-ODS column. All fractions obtained were tested using a microbial growth inhibition test against the S. agalactiae strain (ATCC 12386). Five clinical isolates were used to confirm the action of the fractions with antimicrobial activity, and the bacterial growth curve was determined. Identification of the antimicrobial compounds was carried out through liquid chromatography coupled with mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR). The active compounds found to exhibit antimicrobial activity were Ƴ-glutamyl-S-allyl-cysteine (fraction 18), Ƴ-glutamyl-phenylalanine (fraction 20), and the two stereoisomers (E and Z) of ajoene (fraction 42). The MICs of these fractions were 5.41 mg/ml, 4.60 mg/ml, and 0.16 mg/ml, respectively, and they inhibited the growth of the clinical isolates tested. Antimicrobial compounds from garlic may be a promising source in the search for new drugs against S. agalactiae

High time resolution and high signal-to-noise monitoring of the bacterial growth kinetics in the presence of plasmonic nanoparticles.

BACKGROUND: Emerging concepts for designing innovative drugs (i.e., novel generations of antimicrobials) frequently include nanostructures, new materials, and nanoparticles (NPs). Along with numerous advantages, NPs bring limitations, partly because they can limit the analytical techniques used for their biological and in vivo validation. From that standpoint, designing innovative drug delivery systems requires advancements in the methods used for their testing and investigations. Considering the well-known ability of resazurin-based methods for rapid detection of bacterial metabolisms with very high sensitivity, in this work we report a novel optimization for tracking bacterial growth kinetics in the presence of NPs with specific characteristics, such as specific optical properties. RESULTS: Arginine-functionalized gold composite (HAp/Au/arginine) NPs, used as the NP model for validation of the method, possess plasmonic properties and are characterized by intensive absorption in the UV/vis region with a surface plasmon resonance maximum at 540 nm. Due to the specific optical properties, the NP absorption intensively interferes with the light absorption measured during the evaluation of bacterial growth (optical density; OD600). The results confirm substantial nonspecific interference by NPs in the signal detected during a regular turbidity study used for tracking bacterial growth. Instead, during application of a resazurin-based method (Presto Blue), when a combination of absorption and fluorescence detection is applied, a substantial increase in the signal-to-noise ratio is obtained that leads to the improvement of the accuracy of the measurements as verified in three bacterial strains tested with different growth rates (E. coli, P. aeruginosa, and S. aureus). CONCLUSIONS: Here, we described a novel procedure that enables the kinetics of bacterial growth in the presence of NPs to be followed with high time resolution, high sensitivity, and without sampling during the kinetic study. We showed the applicability of the Presto Blue method for the case of HAp/Au/arginine NPs, which can be extended to various types of metallic NPs with similar characteristics. The method is a very easy, economical, and reliable option for testing NPs designed as novel antimicrobials.

Proteomic analysis of antimicrobial effects of pegylated silver coated carbon nanotubes in Salmonella enterica serovar Typhimurium.

BACKGROUND: Synthesis of silver nano-compounds with enhanced antimicrobial effects is of great interest for the development of new antibacterial agents. Previous studies have reported the antibacterial properties of pegylated silver-coated carbon nanotubes (pSWCNT-Ag) showing less toxicity in human cell lines. However, the mechanism underlining the pSWCNT-Ag as a bactericidal agent remained unfolded. Here we assessed the pSWCNT-Ag effects against foodborne pathogenic bacteria growth and proteome profile changes. RESULTS: Measurements of bioluminescent imaging, optical density, and bacteria colony forming units revealed dose-dependent and stronger bactericidal activity of pSWCNT-Ag than their non-pegylated counterparts (SWCNT-Ag). In ovo administration of pSWCNT-Ag or phosphate-buffered saline resulted in comparable chicken embryo development and growth. The proteomic analysis, using two-dimensional electrophoresis combined with matrix assisted laser desorption/ionization time of flight/time of flight mass spectrometry, was performed on control and surviving Salmonella enterica serovar Typhimurium to pSWCNT-Ag. A total of 15 proteins (ten up-regulated and five down-regulated) differentially expressed proteins were identified. Functional analyses showed significant reduction of proteins associated with biofilm formation, nutrient and energy metabolism, quorum sensing and maintenance of cell structure and cell motility in surviving S. Typhimurium. In contrast, proteins associated with oxygen stress, DNA protection, starvation, membrane rebuilding, and alternative nutrient formation were induced as the compensatory reaction. CONCLUSIONS: This study provides further evidence of the antibacterial effects of pSWCNT-Ag nanocomposites and knowledge of their mechanism of action through various protein changes. The findings may lead to the development of more effective and safe antimicrobial agents.

Inhibition of selected bacterial growth by three hydrocarbons: mathematical evaluation of toxicity using a toxicodynamic equation.

The individual toxicity of different hydrocarbons (naphthalene, cyclododecane and aniline) on the growth of selected bacteria (Pseudomonas sp., Phaeobacter sp. and Leuconostoc mesenteroides) was studied by means of a toxicodynamic model combination of two sigmoid equations (logistic and Weibull). All the toxicological effects on growth parameters and kinetic properties were characterized and the global toxicity of such chemicals was evaluated. It was observed that two kinetic parameters (maximum growth and maximum growth rate) were in almost all cases influenced by the hydrocarbons studied. Aniline was less toxic than cyclododecane and naphthalene. The presented approach is a reasonable starting point for understanding and modeling complete and real assessment of chemical toxic effects on bacterial growths. The values of EC50,τ could be used for a most efficient comparison of the individual toxicity of chemicals.