Gene Networks and Pathways Involved in Escherichia coli Response to Multiple Stressors.

Stress response helps microorganisms survive extreme environmental conditions and host immunity, making them more virulent or drug resistant. Although both reductionist approaches investigating specific genes and systems approaches analyzing individual stress conditions are being used, less is known about gene networks involved in multiple stress responses. Here, using a systems biology approach, we mined hundreds of transcriptomic data sets for key genes and pathways involved in the tolerance of the model microorganism Escherichia coli to multiple stressors. Specifically, we investigated the E. coli K-12 MG1655 transcriptome under five stresses: heat, cold, oxidative stress, nitrosative stress, and antibiotic treatment. Overlaps of transcriptional changes between studies of each stress factor and between different stressors were determined: energy-requiring metabolic pathways, transport, and motility are typically downregulated to conserve energy, while genes related to survival, bona fide stress response, biofilm formation, and DNA repair are mainly upregulated. The transcription of 15 genes with uncharacterized functions is higher in response to multiple stressors, which suggests they may play pivotal roles in stress response. In conclusion, using rank normalization of transcriptomic data, we identified a set of E. coli stress response genes and pathways, which could be potential targets to overcome antibiotic tolerance or multidrug resistance.

Antibacterial and Anti-Inflammatory Activities of Thymus vulgaris Essential Oil Nanoemulsion on Acne Vulgaris.

Antibiotics are frequently used in acne treatment and their prolonged use has led to an emergence of resistance. This study aimed to investigate the use of natural antimicrobials as an alternative therapy. The antimicrobial and anti-inflammatory activities of five commonly used essential oils (EOs) (tea tree, clove, thyme, mentha and basil EOs), and their possible mechanisms of action against Cutibacterium acnes and Staphylococcus epidermidis, were explored. The effect of the most potent EO on membrane permeability was elucidated and its anti-inflammatory action, when formulated as nanoemulsion, was tested in an in vivo acne model. The in vitro studies showed that thyme EO had the most potent antimicrobial and antibiofilm activity, with phenolics and terpenoids as main antimicrobial constituents of EO. Thyme EO affected cell membrane permeability of both bacterial species, evident by the detection of the leakage of intracellular ions and membrane integrity by the leakage of nucleic acids. Morphological alteration in bacterial cells was confirmed by transmission electron microscopy. Thyme EO nanoemulsion led to the suppression of an inflammatory response in acne animal models along with a bacterial load decrease and positive histopathological changes. Collectively, thyme EO nanoemulsion showed potent antimicrobial and anti-inflammatory effects compared to the reference antibiotics, suggesting its effectiveness as a natural alternative in acne treatment.

Immunological characterization of the chemically prepared ghosts of Salmonella Typhimurium as a vaccine candidate.

BACKGROUND: Bacterial ghosts are the evacuated bacterial cellular membranes from most of the genetic and protein contents which preserved their surface characters. Recently, bacterial ghosts exploited for different biomedical applications, for instance, vaccination. The purpose of this study is to measure the immunogenic protective response of bacterial ghosts of Salmonella Typhimurium in animals and to allow future testing this response in humans. The immunologic response was qualitatively, quantitatively, and functionally measured. We have measured the humoral and cellular immune responses, such as immunoglobulins elevation (IgG), increased granulocytes, serum antibacterial activity, clearance of virulence in feces and liver, and the survival rate. RESULTS: The bacterial ghosts' vaccine was able to protect 100% of subcutaneously vaccinated rats and 75% of adjuvant subcutaneously vaccinated rats. The lowest survival rate was in the orally vaccinated group (25%). The maximum level of serum IgG titers, as well as serum and feces bactericidal activity (100% eradication), was exhibited in the subcutaneously vaccinated group with adjuvant vaccines followed by the subcutaneously vaccinated one. Additionally, the highest granulocytes' number was observed in the adjuvant vaccine subcutaneously immunized group. The bacterial load in liver homogenate was eliminated in the subcutaneously vaccinated rats after the virulence challenge. CONCLUSIONS: The bacterial ghosts of Salmonella enterica serovar Typhimurium that prepared by Tween 80 Protocol showed an effective vaccine candidate that protected animals, eliminated the virulence in feces and liver. These findings show that chemically induced bacterial ghosts of Salmonella Typhimurium can be a promising vaccine.

Genomics-guided identification of a conserved CptBA-like toxin-antitoxin system in Acinetobacter baumannii.

Introduction: Toxin-antitoxin (TA) systems are widespread among bacteria, archaea and fungi. They are classified into six types (I-VI) and have recently been proposed as novel drug targets. Objectives: This study aimed to screen the pathogen Acinetobacter baumannii, known for its alarming antimicrobial resistance, for TA systems and identified a CptBA-like type IV TA, one of the least characterized systems. Methods: In silico methods included secondary structure prediction, comparative genomics, multiple sequence alignment, and phylogenetic analysis, while in vitro strategies included plasmid engineering and expression of the TA system in Escherichia coli BL21, growth measurement, and transcription analysis with quantitative reverse-transcription polymerase chain reaction. Results: Comparative genomics demonstrated the distribution of CptBA-like systems among Gram-negative bacteria, while phylogenetic analysis delineated two major groups, in each of which Acinetobacter spp. proteins clustered together. Sequence alignment indicated the conservation of cptA and cptB in 4,732 strains of A. baumannii in the same syntenic order. Using A. baumannii recombinant cptA and cptB, cloned under different promoters, confirmed their TA nature, as cptB expression was able to reverse growth inhibition by CptA in a dose-time dependent manner. Furthermore, transcriptional analysis of cptBA in clinical and standard A. baumannii strains demonstrated the downregulation of this system under oxidative and antibiotic stress. Conclusion: Combining in silico and in vitro studies confirmed the predicted TA nature of a cptBA-like system in A. baumannii . Transcriptional analysis suggests a possible role of cptBA in response to antibiotics and stress factors in A. baumannii, making it a promising drug target.

Salmonella-innovative targeting carrier: Loading with doxorubicin for cancer treatment.

Cell- based targeted delivery is recently gain attention as a promising platform for delivery of anticancer drug in selective and efficient manner. As a new biotechnology platform, bacterial ghosts (BGs) have novel biomedical application as targeted drug delivery system (TDDS). In the current work, Salmonellas' BGs was utilized for the first time as hepatocellular cancer (HCC) in-vitro targeted delivery system. Successful BGs loading and accurate analysis of doxorubicin (DOX) were necessary steps for testing the applicability of DOX loaded BGs in targeting the liver cancer cells. Loading capacity was maximized to reach 27.5 µg/mg (27.5% encapsulation efficiency), by incubation of 10 mg BGs with 1 mg DOX at pH 9 in constant temperature (25 °C) for 10 min. In-vitro release study of DOX loaded BGs showed a sustained release (182 h) obeying Higuchi sustained kinetic release model. The death rate (tested by MTT assay) of HepG2 reached to 64.5% by using of 4 µg/ml, while it was about 51% using the same concentration of the free DOX (P value < 0.0001 One-way ANOVA analysis). The proliferative inhibitory concentration (IC50) of the DOX combined formula was 1.328 µg/ml that was about one third of the IC50 of the free DOX (3.374 µg/ml). Apoptosis analysis (tested by flow-cytometry) showed more accumulation in early apoptosis (8.3%) and late apoptosis/necrosis (91%) by applying 1 µg/ml BGs combined DOX, while 1 µg/ml free DOX showed 33.4% of cells in early apoptosis and 39.3% in late apoptosis/necrosis, (P value˃ 0.05: one-way ANOVA). In conclusion, DOX loaded Salmonellas' BGs are successfully prepared and tested in vivo with promising potential as hepatocellular cancer (HCC) targeted delivery system.

A novel protocol for bacterial ghosts' preparation using tween 80.

Bacterial ghosts (BGs) can be prepared by both genetic and chemical means. Genetic method include using lysis gene E. Chemical method include incubation with numerous agents for a short time at their minimum inhibitory or minimum growth concentrations (MIC or MGC). The aim of this study is to prepare the BGs with a new protocol via exposing the bacterial cells to tween 80 for an extended period of time followed by sudden reduction of the surrounding pH. Salmonella enterica serovar typhimurium ATCC 13311 was used for this purpose. The cells were incubated in 7% v/v tween 80 solution in Muller-Hinton broth for 24 h at 37 °C then pH was decreased to 3.6 by adding lactic acid for one hour. The bacterial pellets were separated by high speed centrifugation, and then washed three times by half normal saline solution. High quality BGs were visualized by scanning electron microscopy (SEM) revealing punctured cells with intact outer shells and at least one intramembranous tunnel. The absence of vital cells was confirmed by subculturing. The release of respective amounts of proteins and DNA is another evidence of ghost's production. In addition, the integrity of cells was proved by visualization of Gram-stained cells using light microscopy. In conclusion, this new protocol is simple, economic and feasible for BGs preparation.