Showing papers on "Escherichia coli published in 2021"

Bacteria-derived long chain fatty acid exhibits anti-inflammatory properties in colitis

Abstract: Objective Data from clinical research suggest that certain probiotic bacterial strains have the potential to modulate colonic inflammation. Nonetheless, these data differ between studies due to the probiotic bacterial strains used and the poor knowledge of their mechanisms of action. Design By mass-spectrometry, we identified and quantified free long chain fatty acids (LCFAs) in probiotics and assessed the effect of one of them in mouse colitis. Results Among all the LCFAs quantified by mass spectrometry in Escherichia coli Nissle 1917 (EcN), a probiotic used for the treatment of multiple intestinal disorders, the concentration of 3-hydroxyoctadecaenoic acid (C18-3OH) was increased in EcN compared with other E. coli strains tested. Oral administration of C18-3OH decreased colitis induced by dextran sulfate sodium in mice. To determine whether other bacteria composing the microbiota are able to produce C18-3OH, we targeted the gut microbiota of mice with prebiotic fructooligosaccharides (FOS). The anti-inflammatory properties of FOS were associated with an increase in colonic C18-3OH concentration. Microbiota analyses revealed that the concentration of C18-3OH was correlated with an increase in the abundance in Allobaculum, Holdemanella and Parabacteroides. In culture, Holdemanella biformis produced high concentration of C18-3OH. Finally, using TR-FRET binding assay and gene expression analysis, we demonstrated that the C18-3OH is an agonist of peroxisome proliferator activated receptor gamma. Conclusion The production of C18-3OH by bacteria could be one of the mechanisms implicated in the anti-inflammatory properties of probiotics. The production of LCFA-3OH by bacteria could be implicated in the microbiota/host interactions.

The Desk Encyclopedia Of Microbiology

Abstract: Adhesion, Bacterial Agrobacterium and Plant Cell Transformation Antibiotic Resistance in Bacteria Antifungal Agents Antisense RNAs Antiviral Agents Archaea Attenuation, Transcriptional Bacillus subtilis, Genetics Bacteriophages Biocides: Nonpublic Health, Nonagricultural Antimicrobials Biofilms and Biofouling Biological Warfare Bioluminescence, Microbial Bioreactors Bioremedation Biosensors Cell Membrane: Structure and Function Cell Walls, Bacterial Chemotaxis Chromosome, Bacterial Conjugation, Bacterial Crystalline Bacterial Cell Surface Layers (S Layers) Culture Collections and Their Databases Developmental Processes in Bacteria Diversity, Microbial DNA Repair DNA Replication DNA Restriction and Modification DNA Sequencing and Genomics Ecology, Microbial Emerging Infections Energy Transduction Processes Enteropathogenic Bacteria Escherichia coli and Salmonella, Genetics Exotoxins Extremophiles Fimbriae, Pili Flagella Foodborne Illnesses Fungal Infections, Cutaneous Fungal Infections, Systemic Gastrointestinal Microbiology Genetically Modified Organisms: Guidelines and Regulations for Research Genomes, Mapping of Bacterial Germfree Animal Techniques Gram-Negative Anaerobic Pathogens Gram-Negative Cocci, Pathogenic Heat Stress Horizontal Transfer of Genes Between Microorganisms Human Immunodeficiency Virus (HIV) Identification of Bacteria, Computerized Industrial Fermentation Processes Insects' Symbiotic Microorganisms Iron Metabolism Lipopolysaccharides Methanogenesis Methylotrophy Nitrogen Cycle Nitrogen Fixation Nodule Formation in Legumes Nutrition of Microorganisms Oral Microbiology Osmotic Stress Outer Membrane, Gram-Negative Bacteria Oxidative Stress pH Stress Plant Pathogens Plasmids, Bacterial Polymerase Chain Reaction (PCR) Prions Protein Secretion Quorum-Sensing in Gram-Negative Bacteria Recombinant DNA, Basic Procedures Sexually Transmitted Diseases Skin Microbiology Soil Microbiology SOS Response Space Flight, Effects on Microorganisms Sporulation Starvation, Bacterial Strain improvement Sulfur Cycle Transcriptional Regulation Transduction: Host DNA Transfer by Bacteriophages Transformation, Genetic Transposable Elements Two-Component Systems Vaccines, Bacterial Vaccines, Viral Viruses Viruses, Emerging Yeasts

Plasmid- and strain-specific factors drive variation in ESBL-plasmid spread in vitro and in vivo.

Abstract: Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data.

Anti-cancer and anti-inflammatory effects elicited by short chain fatty acids produced by Escherichia coli isolated from healthy human gut microbiota.

Abstract: Extracellular metabolites of short chain fatty acids (SCFA) excreted by gut microbiota have been reported to play an important role in the regulation of intestinal homeostasis. Apart from supplying energy, SCFA also elicit immune stimulation in animal and human cells. Therefore, an attempt was conducted to isolate SCFA producing bacteria from healthy human microbiota. The anti-cancer and anti-inflammatory effects of extracellular metabolites and individual SFCA were further investigated by using breast, colon cancer and macrophage cells. Toxin, inflammatory and anti-inflammatory cytokine gene expressions were investigated by RT-qPCR analyses in this study. Escherichia coli KUB-36 was selected in this study since it has the capability to produce seven SCFA extracellularly. It produced acetic acid as the main SCFA. It is a non-exotoxin producer and hence, it is a safe gut microbiota. The IC50 values indicated that the E. coli KUB-36 metabolites treatment elicited more potent cytotoxicity effect on MCF7 breast cancer cell as compared to colon cancer and leukemia cancer cells but exhibited little cytotoxic effects on normal breast cell. Furthermore, E. coli KUB-36 metabolites and individual SCFA could affect inflammatory responses in lipopolysaccharide-induced THP-1 macrophage cells since they suppressed inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α well as compared to the control, whilst inducing anti-inflammatory cytokine IL-10 expression. SCFA producing E. coli KUB-36 possessed vast potential as a beneficial gut microbe since it is a non-exotoxin producer that exhibited beneficial cytotoxic effects on cancer cells and elicited anti-inflammatory activity simultaneously. However, the probiotic characteristic of E. coli KUB-36 should be further elucidated using in vivo animal models.

Shiga Toxin-Producing Escherichia coli Transmission via Fecal Microbiota Transplant.

Abstract: Fecal microbiota transplantation (FMT) is recommended therapy for multiply recurrent Clostridioides difficile infection. We report adverse events in 7 patients who received FMT from a stool donor who was colonized with Shiga toxin-producing Escherichia coli (STEC). No patients died of FMT-transmitted STEC. Improved screening can likely avoid future transmission.

Identification of mucin degraders of the human gut microbiota

Abstract: Mucins are large glycoproteins consisting of approximately 80% of hetero-oligosaccharides. Gut mucin degraders of healthy subjects were investigated, through a culture dependent and independent approach. The faeces of five healthy adults were subjected to three steps of anaerobic enrichment in a medium with sole mucins as carbon and nitrogen sources. The bacterial community was compared before and after the enrichment by 16S rRNA gene profiling. Bacteria capable of fermenting sugars, such as Anaerotruncus, Holdemania, and Enterococcaceae likely took advantage of the carbohydrate chains. Escherichia coli and Enterobacteriaceae, Peptococcales, the Coriobacteriale Eggerthella, and a variety of Clostridia such as Oscillospiraceae, Anaerotruncus, and Lachnoclostridium, significantly increased and likely participated to the degradation of the protein backbone of mucin. The affinity of E. coli and Enterobacteriaceae for mucin may facilitate the access to the gut mucosa, promoting gut barrier damage and triggering systemic inflammatory responses. Only three species of strict anaerobes able to grow on mucin were isolated from the enrichments of five different microbiota: Clostridium disporicum, Clostridium tertium, and Paraclostridium benzoelyticum. The limited number of species isolated confirms that in the gut the degradation of these glycoproteins results from cooperation and cross-feeding among several species exhibiting different metabolic capabilities.

Purification and characterization of the receptor-binding domain of SARS-CoV-2 spike protein from Escherichia coli

Abstract: SARS-CoV-2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID-19. Spike protein of SARS-CoV-2 mediates viral entry into host cells by binding ACE2 through the receptor-binding domain (RBD). RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines. RBD expressed in mammalian cells suffers from low expression yield and high cost. E. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. However, RBD expressed by E. coli (RBD-1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. In the present study, RBD-1 was expressed by E. coli and purified by a Ni Sepharose Fast Flow column. RBD-1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD-2). The secondary structure and tertiary structure of RBD-1 were largely maintained without glycosylation. In particular, the major beta-sheet content of RBD-1 was almost unaltered. RBD-1 could strongly bind ACE2 with a dissociation constant (K-D) of 2.98 x 10(-8) M. Thus, RBD-1 was expected to apply in the vaccine development, screening drugs and virus test kit.

Antibiotic resistance plasmid composition and architecture in Escherichia coli isolates from meat.

Abstract: Resistance plasmids play a crucial role in the transfer of antimicrobial resistance from the veterinary sector to human healthcare. In this study plasmids from foodborne Escherichia coli isolates with a known (ES)BL or tetracycline resistance were sequenced entirely with short- and long-read technologies to obtain insight into their composition and to identify driving factors for spreading. Resistant foodborne E. coli isolates often contained several plasmids coding for resistance to various antimicrobials. Most plasmids were large and contained multiple resistance genes in addition to the selected resistance gene. The majority of plasmids belonged to the IncI, IncF and IncX incompatibility groups. Conserved and variable regions could be distinguished in each of the plasmid groups. Clusters containing resistance genes were located in the variable regions. Tetracycline and (extended spectrum) beta-lactamase resistance genes were each situated in separate clusters, but sulphonamide, macrolide and aminoglycoside formed one cluster and lincosamide and aminoglycoside another. In most plasmids, addiction systems were found to maintain presence in the cell.

Antibacterial characteristics and mechanisms of action of Aronia melanocarpa anthocyanins against Escherichia coli

Abstract: This study aimed to evaluate the antibacterial activity and mechanism of action of Aronia melanocarpa anthocyanins (AMAs) against Escherichia coli. AMAs displayed strong antibacterial activity against Escherichia coli. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of AMAs against E. coli was 0.625 mg/mL and 1.25 mg/mL, respectively. Analysis of alkaline phosphatase activity, potassium ion leakage, absorption material release at 260 nm, and change in soluble protein content revealed that AMAs could destroy the cell wall and membrane structure of E. coli. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) further confirmed the cell wall and cell membrane damage induced by AMAs. In addition, the interaction between AMAs and DNA was elucidated through agarose gel retardation and fluorescence spectroscopy. In summary, the present results indicate that AMAs kill E. coli by attacking multiple targets in the bacterial cell, which could favour the application of AMAs as natural food preservatives.

Virulence Determinants and Multidrug Resistance of Escherichia coli Isolated from Migratory Birds

Abstract: Migratory birds are carriers of multidrug resistant pathogenic Escherichia coli. However, their roles in the dissemination of these resistant pathogens are still being neglected in Bangladesh. The present study was therefore carried out to detect multidrug resistant E. coli. In addition, these isolates were also screened for the presence of avian pathogenic E. coli (APEC)-associated virulence genes. A total of 66 fecal matter samples of migratory birds were screened. E. coli were isolated and identified by culturing and biochemical tests followed by polymerase chain reaction (PCR). APEC-associated virulence genes were detected by PCR. Disk diffusion assays were employed to investigate antibiogram profiles. Bivariate analysis was performed to assess correlations in resistance patterns between antimicrobials and to assess associations between virulence genes of E. coli. Among the 66 samples assessed by PCR, 55 (83.33%) were found positive for E. coli. Of these 55 isolates, the APEC-associated virulence gene fimC was detected in 67.27% of the isolates, which was significantly higher than in the cases of iucD (29.09%) and papC (5.45%) genes. In addition, three isolates were found positive for all three virulence genes, while 23 and 12 isolates were positive for one and two virulence genes respectively. In the bivariate analysis, significant associations were detected between fimC and iucD virulence genes. Using the antibiogram, all E. coli isolates were found to be multidrug resistant (MDR). The isolates exhibited 100% resistance against ampicillin and erythromycin in addition to varying percentages of resistance against streptomycin, tetracycline, ciprofloxacin, and chloramphenicol. Highly positive correlations between tetracycline and ciprofloxacin, chloramphenicol and ciprofloxacin, chloramphenicol and tetracycline were observed by bivariate analysis. To the best of our knowledge, this is the first study that reports APEC-associated virulence genes of MDR E. coli from migratory birds in Bangladesh. Results indicate that migratory birds are reservoirs of MDR E. coli isolates carrying APEC-associated virulence genes, which can seriously contribute to the development of human and animal diseases.

Bacteriophages of Shiga Toxin-Producing Escherichia coli and Their Contribution to Pathogenicity.

Abstract: Shiga toxins (Stx) of Shiga toxin-producing Escherichia coli (STEC) are generally encoded in the genome of lambdoid bacteriophages, which spend the most time of their life cycle integrated as prophages in specific sites of the bacterial chromosome. Upon spontaneous induction or induction by chemical or physical stimuli, the stx genes are co-transcribed together with the late phase genes of the prophages. After being assembled in the cytoplasm, and after host cell lysis, mature bacteriophage particles are released into the environment, together with Stx. As members of the group of lambdoid phages, Stx phages share many genetic features with the archetypical temperate phage Lambda, but are heterogeneous in their DNA sequences due to frequent recombination events. In addition to Stx phages, the genome of pathogenic STEC bacteria may contain numerous prophages, which are either cryptic or functional. These prophages may carry foreign genes, some of them related to virulence, besides those necessary for the phage life cycle. Since the production of one or more Stx is considered the major pathogenicity factor of STEC, we aim to highlight the new insights on the contribution of Stx phages and other STEC phages to pathogenicity.

Synthesis, antimicrobial evaluation, DNA gyrase inhibition, and in silico pharmacokinetic studies of novel quinoline derivatives.

Abstract: Herein, we report the synthesis and in vitro antimicrobial evaluation of novel quinoline derivatives as DNA gyrase inhibitors. The preliminary antimicrobial activity was assessed against a panel of pathogenic microbes including Gram-positive bacteria (Streptococcus pneumoniae and Bacillus subtilis), Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), and fungal strains (Aspergillus fumigatus, Syncephalastrum racemosum, Geotrichum candidum, and Candida albicans). Compounds that revealed the best activity were subjected to further biological studies to determine their minimum inhibitory concentrations (MICs) against the selected pathogens as well as their in vitro activity against the E. coli DNA gyrase, to realize whether their antimicrobial action is mediated via inhibition of this enzyme. Four of the new derivatives (14, 17, 20, and 23) demonstrated a relatively potent antimicrobial activity with MIC values in the range of 0.66-5.29 μg/ml. Among them, compound 14 exhibited a particularly potent broad-spectrum antimicrobial activity against most of the tested strains of bacteria and fungi, with MIC values in the range of 0.66-3.98 μg/ml. A subsequent in vitro investigation against the bacterial DNA gyrase target enzyme revealed a significant potent inhibitory activity of quinoline derivative 14, which can be observed from its IC50 value (3.39 μM). Also, a molecular docking study of the most active compounds was carried out to explore the binding affinity of the new ligands toward the active site of DNA gyrase enzyme as a proposed target of their activity. Furthermore, the ADMET profiles of the most highly effective derivatives were analyzed to evaluate their potentials to be developed as good drug candidates.

Improved broad-spectrum antibiotics against Gram-negative pathogens via darobactin biosynthetic pathway engineering

Abstract: The development of new antibiotics is imperative to fight increasing mortality rates connected to infections caused by multidrug-resistant (MDR) bacteria. In this context, Gram-negative pathogens listed in the WHO priority list are particularly problematic. Darobactin is a ribosomally produced and post-translationally modified bicyclic heptapeptide antibiotic selectively killing Gram-negative bacteria by targeting the outer membrane protein BamA. The native darobactin A producer Photorhabdus khanii HGB1456 shows very limited production under laboratory cultivation conditions. Herein, we present the design and heterologous expression of a synthetically engineered darobactin biosynthetic gene cluster (BGC) in Escherichia coli to reach an average darobactin A production titre of 13.4 mg L-1. Rational design of darA variants, encoding the darobactin precursor peptide with altered core sequences, resulted in the production of 13 new 'non-natural' darobactin derivatives and 4 previously hypothetical natural darobactins. One of the non-natural compounds, darobactin 9, was more potent than darobactin A, and showed significantly improved activity especially against Pseudomonas aeruginosa (0.125 μg mL-1) and Acinetobacter baumannii (1-2 μg mL-1). Importantly, it also displayed superior activity against MDR clinical isolates of E. coli (1-2 μg mL-1) and Klebsiella pneumoniae (1-4 μg mL-1). Independent deletions of genes from the darobactin BGC showed that only darA and darE, encoding a radical forming S-adenosyl-l-methionine-dependent enzyme, are required for darobactin formation. Co-expression of two additional genes associated with the BGCs in hypothetical producer strains identified a proteolytic detoxification mechanism as a potential self-resistance strategy in native producers. Taken together, we describe a versatile heterologous darobactin platform allowing the production of unprecedented active derivatives in good yields, and we provide first experimental evidence for darobactin biosynthesis processes.

Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

Abstract: Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (P T7 lac , Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1' and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

Bacteria-Anchoring Hybrid Liposome Capable of Absorbing Multiple Toxins for Antivirulence Therapy of Escherichia coli Infection.

Abstract: Antivirulence therapy by cell membrane coated nanoparticles has shown promise against bacterial infections. However, current approaches remain unsatisfactory when facing Escherichia coli (E. coli) infections, since the E. coli secretes multiple bacterial toxins including endotoxins and exotoxins that are challenging to eliminate simultaneously. What is worse, the absorptive scavengers normally rely on random contact of the diffuse toxins, which is not efficient. For the current cell membrane coated platform, the single type of cell membrane cannot fully meet the detoxing requirement facing multiple toxins. To address these problems, a polymyxin B (PMB)-modified, red blood cell (RBC)-mimetic hybrid liposome (P-RL) was developed. The P-RL was fabricated succinctly through fusion of PMB-modified lipids and the RBC membranes. By the strong interaction between PMB and the E. coli membrane, P-RL could attach and anchor to the E. coli; attributed to the fused RBC membrane and modified PMB, the P-RL could then efficiently neutralize both endotoxins and exotoxins from the toxin fountainhead. In vitro and in vivo results demonstrated the P-RL had a significant anchoring effect to E. coli. Moreover, compared with the existing RBC vesicles or PMB-modified liposomes, P-RL exhibited a superior therapeutic effect against RBC hemolysis, macrophage activation, and a mixed-toxin infection in mice. Potently, P-RL could inhibit E. coli O157:H7-induced skin damage, intestinal infection, and mouse death. Overall, the P-RL could potentially improve the detoxing efficiency and markedly expand the detoxification spectrum of current antivirulence systems, which provides different insights into drug-resistant E. coli treatment.

Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning.

Abstract: Bacteria can cause diseases, but they also battle their own microscopic enemies: a group of viruses known as bacteriophages. For instance, the T7 bacteriophage preys on various strains of Escherichia coli, a type of bacteria often found in the human gut. While many E. coli strains are inoffensive or even beneficial to human health, some can be deadly. Finding a way to kill harmful strains while sparing the helpful ones would be a helpful addition to the medicine toolkit. Bacteriophages identify and interact with their specific target through a structure known as the receptor binding protein, or RBP. However, it is still unclear exactly how RBP helps the viruses recognize which type of bacteria to infect. Here, Huss et al. set to map out and modify this structure in T7 bacteriophage so the virus is more efficient and specific about which strain of E. coli it kills. First, the role of each building block in the tip of RBP was meticulously dissected; this generated the knowledge required to genetically engineer a large number of different T7 bacteriophages, each with a slightly variation in their RBP. These viruses were then exposed to various strains of bacteria. Monitoring the bacteriophages that survived and multiplied the most after infecting different strains of E. coli revealed which RBP building blocks are important for efficiency and specificity. This was then confirmed by engineering highly active T7 bacteriophage variants against an E. coli strain that causes urinary tract infections. These findings demonstrate that even small changes to the bacteriophages can make a big difference to their ability to infect their preys. The approaches developed by Huss et al. help to understand exactly how the RBP allows a virus to infect a specific type of bacteria; this could one day pave the way for new therapies that harness those viruses to fight increasingly resistant bacterial infections.

Outer Membrane Vesicles Derived from Klebsiella pneumoniae Are a Driving Force for Horizontal Gene Transfer.

Abstract: Gram-negative bacteria release Outer Membrane Vesicles (OMVs) into the extracellular environment. Recent studies recognized these vesicles as vectors to horizontal gene transfer; however, the parameters that mediate OMVs transfer within bacterial communities remain unclear. The present study highlights for the first time the transfer of plasmids containing resistance genes via OMVs derived from Klebsiella pneumoniae (K. pneumoniae). This mechanism confers DNA protection, it is plasmid copy number dependent with a ratio of 3.6 times among high copy number plasmid (pGR) versus low copy number plasmid (PRM), and the transformation efficiency was 3.6 times greater. Therefore, the DNA amount in the vesicular lumen and the efficacy of horizontal gene transfer was strictly dependent on the identity of the plasmid. Moreover, the role of K. pneumoniae-OMVs in interspecies transfer was described. The transfer ability was not related to the phylogenetic characteristics between the donor and the recipient species. K. pneumoniae-OMVs transferred plasmid to Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Burkholderia cepacia. These findings address the pivotal role of K. pneumoniae-OMVs as vectors for antimicrobial resistance genes spread, contributing to the development of antibiotic resistance in the microbial communities.

β-lactam resistance associated with β-lactamase production and porin alteration in clinical isolates of E. coli and K. pneumoniae.

Abstract: β-lactam resistance represents a worldwide problem and a serious challenge for antimicrobial treatment. Hence this research was conducted to recognize several mechanisms mediating β-lactam resistance in E. coli and K. pneumoniae clinical isolates collected from Mansoura University hospitals, Egypt. A total of 80 isolates, 45 E. coli and 35 K. pneumoniae isolates, were collected and their antibiotic susceptibility was determined by the Disc diffusion method followed by phenotypic and genotypic detection of extended-spectrum β-lactamases (ESBLs), AmpC β-lactamase, carbapenemase enzymes. The outer membrane protein porins of all isolates were analyzed and their genes were examined using gene amplification and sequencing. Also, the resistance to complement-mediated serum killing was estimated. A significant percentage of isolates (93.8%) were multidrug resistance and showed an elevated resistance to β-lactam antibiotics. The presence of either ESBL or AmpC enzymes was high among isolates (83.75%). Also, 60% of the isolated strains were carbapenemase producers. The most frequently detected gene of ESBL among all tested isolates was blaCTX-M-15 (86.3%) followed by blaTEM-1 (81.3%) and blaSHV-1 (35%) while the Amp-C gene was present in 83.75%. For carbapenemase-producing isolates, blaNDM1 was the most common (60%) followed by blaVIM-1 (35%) and blaOXA-48 (13.8%). Besides, 73.3% and 40% of E. coli and K. pneumoniae isolates respectively were serum resistant. Outer membrane protein analysis showed that 93.3% of E. coli and 95.7% of K. pneumoniae isolates lost their porins or showed modified porins. Furthermore, sequence analysis of tested porin genes in some isolates revealed the presence of frameshift mutations that produced truncated proteins of smaller size. β-lactam resistance in K. pneumoniae and E. coli isolates in our hospitals is due to a combination of β-lactamase activity and porin loss/alteration. Hence more restrictions should be applied on β-lactams usage to decrease the emergence of resistant strains.

Novel surface plasmon resonance biosensor that uses full-length Det7 phage tail protein for rapid and selective detection of Salmonella enterica serovar Typhimurium.

Abstract: We report a novel surface plasmon resonance (SPR) biosensor that uses the full-length Det7 phage tail protein (Det7T) to rapidly and selectively detect Salmonella enterica serovar Typhimurium (S. Typhimurium). Det7T, which was obtained using recombinant protein expression and purification in Escherichia coli, demonstrated a size of ∼75 kDa upon SDS-PAGE and was homotrimeric in its native structure. Microagglutination and transmission electron microscopy (TEM) data revealed that the protein specifically bound to the host, S. Typhimurium, but not to nonhost E. coli K-12 cells. The observed protein agglutination occurred over a concentration range of 0.8-24.6 µg/mL. The Det7T proteins were immobilized on gold-coated surfaces using amine-coupling to generate a novel Det7T-functionalized SPR biosensor, wherein the specific binding of these proteins with bacteria was detected by SPR. We observed rapid detection of (∼20 Min) and typical binding kinetics with S. Typhimurium in the range of 5 × 104 -5 × 107 CFU/mL, but not with E. coli at any tested concentration, indicating that the sensor exhibited recognition specificity. Similar binding was observed with 10% apple juice spiked with S. Typhimurium, suggesting that this strategy provides promise for the rapid, real-time, and selective monitoring of target microorganisms in the environment, and thus has great potential for supporting health by enabling early disease prevention.

Pathway Optimization of 2'-Fucosyllactose Production in Engineered Escherichia coli

Abstract: 2'-Fucosyllactose (2'-FL), one of the most valuable oligosaccharides in human milk, is used as an emerging food ingredient in the nutraceutical and food industries due to its numerous health benefits. Herein, the de novo and salvage pathways for GDP-fucose synthesis were engineered and optimized in Escherichia coli BL21 (DE3) to improve the production of 2'-FL. The de novo pathway genes encoding phosphomannomutase (ManB), mannose-1-phosphate guanyltransferase (ManC), GDP-d-mannose-4,6-dehydratase (Gmd), and GDP-l-fucose synthase (WcaG) combined with the gene from the salvage pathway encoding fucose kinase/fucose-1-phosphate guanylyltransferase (Fkp) were reconstructed in two vectors to evaluate the GDP-fucose biosynthesis. Then, the fucT2 gene, encoding α1,2-fucosyltransferase, was introduced into the GDP-fucose-overproducing strains to realize 2'-FL biosynthesis. Furthermore, the genes in bypass pathways, including lacZ, fucI, fucK, and wcaJ, were inactivated to improve 2'-FL production. In addition, the two GDP-fucose synthesis pathways, along with fucT2, were transcriptionally fine-tuned to efficiently increase 2'-FL production. The final metabolically engineered E. coli produced 2.62 and 14.1 g/L in shake-flask and fed-batch cultivations, respectively.

Purification and antibacterial properties of a novel bacteriocin against Escherichia coli from Bacillus subtilis isolated from blueberry ferments

Abstract: The novel bacteriocin JS17 was obtained from Bacillus subtilis JSX-5, isolated from blueberry (Vaccinium uliginosum) ferments JS17 was purified by AKTA Purifier, semi-preparative reversed-phase high performance liquid chromatography, and nano liquid chromatography coupled with tandem mass spectrometry, in succession Its molecular mass was 65237 Da and its amino acid sequence was identified as L-F-R-A-F A homology BLAST search confirmed JS17 as a novel bacteriocin JS17 was found to have an extensive antibacterial activity spectrum against both Gram-positive and Gram-negative bacteria JS17 showed heat tolerance, and it was highly stable over pH 2–10 and was sensitive to proteinase K and pepsin This indicates that JS17 exhibits protein properties and has application potential under high temperature and acid-base conditions The minimum inhibitory concentration and minimum bactericide concentration of JS17 against Escherichia coli were 1056 μg/mL and 2203 μg/mL, respectively, which is lower than that of most previously described bacteriocins Furthermore, scanning electron microscopy showed that JS17 bactericidally destroyed the cell membrane integrity of E coli, resulting in cell dissolution and impaired membrane permeability This study first purified B subtilis bacteriocin from fruit ferments, suggesting the potential for using JS17 as a food bio-preservative