A novel approach to cell immobilization with filamentous Escherichia coli

Summary The presence of 18-crown-6 in growth medium produces filamentous Escherichia coli of up to 200 times the normal cell length. The immobilization of these filamentous cells by confinement enables a choice of membranes of larger pore size which in turn speeds up the flow rate of the system. A novel approach of using filamentous cells to facilitate cell immobilization by confinement is proposed.     

Medicinal plant extracts variously modulate susceptibility of Escherichia coli to different antibiotics

Antioxidant activity of green and black tea and extracts of medicinal plants and their ability to modulate antibiotic susceptibility in Escherichia coli were studied. Among a number of extracts tested the maximal capacity to scavenge DPPH radicals and chelate iron in chemical tests was found in green and black tea, Arctostaphylos uva-ursi and Vaccinium vitis-idaea. These extracts contained high level of polyphenols and in aerobic conditions exhibited prooxidant features, producing H₂O₂ and inducing expression of the katG gene encoding catalase HPI in E. coli cells. A good correlation between the polyphenol content and the ability of extracts to protect bacteria against peroxide stress was observed (r = 0.88). Polyphenol-rich extracts and iron chelators demonstrated the highest modulating effect on the antibiotic susceptibility by changing the time period before lysis started and by influencing the colony-forming ability of bacteria. The direction of the modulating effect was dependent on nature of antibiotic applied: under treatment with ciprofloxacin and ampicillin the extracts predominantly provided protective effects, while under treatment with kanamycin a bactericidal action was enhanced. Mechanism of modulating action of extracts on bacterial antibiotic susceptibility probably involves antioxidant, preferentially iron-chelating, or prooxidant properties of polyphenols.     

Frequency of isolation and susceptibility to antibiotics of Escherichia coli strains isolated from blood

The aim of our study was the analysis of Escherichia coli strains obtained from patients of University Hospital No 1 of dr A. Jurasz Collegium Medicum of L. Rydygier in Bydgoszcz Nicolaus Copernicus University in Toruń and State Infectious Diseases Observatory Hospital of T. Browicz in Bydgoszcz, between 2007 and 2010. Among all microorganisms isolated from blood was 8.7% E. coli strains and 45.1% of all rods from Enterobacteriaceae family. Number of E. coli isolations from positive blood samples was: 64 in 2007, 69 in 2008, 77 in 2009 and 26 in the first half of 2010 year. The highest percentage of E. coli strains were obtained from patients of the Transplantology and Surgery Clinic (16.1%), the Nephrology and Internal Diseases Clinic with the Dialysis Centre (14.0%), the Pediatric, Hematology and Oncology Clinic (13.6%) and the Anesthesiology and Intensive Care Clinic (13.6%). All analysed strains were susceptible to carbapenems, amikacin and tygecycline. The highest percentage of resistant strains were observed to ampicillin (70.7%), piperacillin (43.9%), tetracycline (42.8%) and doxycycline (38.8%). During four years of study 4 (6.3%), one, three and two E. coli strains with ESBL were isolated, respectively.     

Escherichia coli murein-DD-endopeptidase insensitive to beta-lactam antibiotics.

A novel endopeptidase degrading the peptide cross-links in sacculi has been isolated from Escherichia coli and purified to homogeneity. The enzyme has a molecular weight of 30,000 and, in contrast to already known enzymes of similar specificity, remains fully active in the presence of beta-lactam antibiotics. In addition, it is exceptional in being inhibited by single-stranded deoxyribonucleic acid and by some polynucleotides. The possible role of the enzyme in cell division is discussed.     

A quantitative approach to catabolite repression in Escherichia coli

A dynamic mathematical model was developed to describe the uptake of various carbohydrates (glucose, lactose, glycerol, sucrose, and galactose) in Escherichia coli. For validation a number of isogenic strains with defined mutations were used. By considering metabolic reactions as well as signal transduction processes influencing the relevant pathways, we were able to describe quantitatively the phenomenon of catabolite repression in E. coli. We verified model predictions by measuring time courses of several extra- and intracellular components such as glycolytic intermediates, EII-ACrr phosphorylation level, both LacZ and PtsG concentrations, and total cAMP concentrations under various growth conditions. The entire data base consists of 18 experiments performed with nine different strains. The model describes the expression of 17 key enzymes, 38 enzymatic reactions, and the dynamic behavior of more than 50 metabolites. The different phenomena affecting the phosphorylation level of EIIACrr, the key regulation molecule for inducer exclusion and catabolite repression in enteric bacteria, can now be explained quantitatively.     

A genome-wide association analysis for susceptibility of pigs to enterotoxigenic Escherichia coli F41

Enterotoxigenic Escherichia coli (ETEC) is a type of pathogenic bacteria that cause diarrhea in piglets through colonizing pig small intestine epithelial cells by their surface fimbriae. Different fimbriae type of ETEC including F4, F18, K99 and F41 have been isolated from diarrheal pigs. In this study, we performed a genome-wide association study to map the loci associated with the susceptibility of pigs to ETEC F41 using 39454 single nucleotide polymorphisms (SNPs) in 667 F₂ pigs from a White Duroc×Erhualian F₂ cross. The most significant SNP (ALGA0022658, P=5.59×10⁻¹³) located at 6.95 Mb on chromosome 4. ALGA0022658 was in high linkage disequilibrium (r²>0.5) with surrounding SNPs that span a 1.21 Mb interval. Within this 1.21 Mb region, we investigated ZFAT as a positional candidate gene. We re-sequenced cDNA of ZFAT in four pigs with different susceptibility phenotypes, and identified seven coding variants. We genotyped these seven variants in 287 unrelated pigs from 15 diverse breeds that were measured with ETEC F41 susceptibility phenotype. Five variants showed nominal significant association (P<0.05) with ETEC F41 susceptibility phenotype in International commercial pigs. This study provided refined region associated with susceptibility of pigs to ETEC F41 than that reported previously. Further works are needed to uncover the underlying causal mutation(s).     

A novel and efficient approach for imparting magnetic susceptibility to lignocellulosic fibers

We have imparted magnetic susceptibility to lignocellulosic fibers by adding iron powder in a heterogeneous manner to the fibers during hydrogen peroxide bleaching chemistry. We have therefore generated carboxylic acid groups in the fibers by deliberately inducing cellulose degradation through Fenton catalysis of the hydrogen peroxide during the chemical oxidation process at a specified level of iron. The iron particles consequently have an exposed layer of iron oxide that allows ionic neutralization of the negatively charged fiber acid groups. After removal of non-attached, excess iron, these fibers have been cast into two-dimensional sheets with two different original iron concentrations and tested for physical and chemical properties. Physical tests included tensile, zero-span tensile, caliper, and surface resistivity. Chemical tests included surface charge, lignin content (kappa) and viscosity. SEM and ICP were also conducted. Remarkably, the magnetically susceptible sheets with incorporated iron were able to retain a tensile strength similar to the unbleached sheets despite attenuation in fiber strength. This is likely due to a chemical refining phenomenon which allowed for increased fiber–fiber bonding. The introduction of the retained iron also significantly alters the surface resistivity of the paper sheets. Such fibers may have a use in applications where charge conduction or dispersion is necessary.     

Tn5 insertion in the polynucleotide phosphorylase (pnp) gene in Escherichia coli increases susceptibility to antibiotics.

A Tn5 insertional mutation on the Escherichia coli chromosome which caused a severalfold increase in susceptibility to structurally and functionally diverse antibiotics was found to map within the gene for polynucleotide phosphorylase (pnp) and to inactivate this enzyme, which is involved in RNA breakdown. The mutation also decreased the growth rate 10 to 25% and increased the rate of tetracycline uptake about 30%. The hypersensitivity due to the insertion was only partially complemented by a cloned pnp gene.     

A conspicuous adaptability to antibiotics in the Escherichia coli mutator strain, dnaQ49

By repeating the cycle of mutagenesis and selection, the Escherichia coli dnaQ49 mutator acquired high level resistance to ampicillin (30,000 micrograms ml-1), streptomycin (26,000 micrograms ml-1) and ofloxacin (3000 micrograms ml-1). Under the strong pressure of ofloxacin, dnaQ49 also followed the history of mutations in the gyrase and topoisomerase i.v. genes previously observed in clinical isolates of quinolone-resistant E. coli. The results of these in vitro experiments suggest that naturally existing mutators may participate in the rapid acquisition of resistance to various antibiotics in patients. A possible mechanism for the occurrence of this adaptability is discussed with special reference to the property of mutagenesis accompanying DNA replication.     

New approach for specific determination of antibiotics by use of luminescent Escherichia coli and immune serum

This paper describes a possible application of luminescent Escherichia coli activated by blood serum for high-sensitivity and high-specificity assays of antibiotics in solutions. Antibiotics inhibited luminescence of a genetically engineered E. coli strain; the system sensitivity to some antibiotics grew notably after the cells had been preactivated by blood serum. The highest level of sensitivity (2.8 +/- 0.6 ng/ml) of luminescent cells was obtained for aminoglycoside antibiotics (gentamicin and streptomycin). It is feasible to create the specific biosensor for antibiotics on the basis of bioluminescent E. coli strains by applying sera containing antibodies against the antibiotic under assay. The presence of antibodies specific for gentamicin in serum affects inhibition of luminescent cells by gentamicin but not inhibition by other antibiotics.     

Fast and reliable determination of Escherichia coli susceptibility to antibiotics: Infrared microscopy in tandem with machine learning algorithms

Antimicrobial drugs have an important role in controlling bacterial infectious diseases. However, the increasing resistance of bacteria to antibiotics has become a global health care problem. Rapid determination of antimicrobial susceptibility of clinical isolates is often crucial for the optimal antimicrobial therapy. The conventional methods used in medical centers for susceptibility testing are time‐consuming (>2 days). Two bacterial culture steps are needed, the first is used to grow the bacteria from urine on agar plates to determine the species of the bacteria (~24 hours). The second culture is used to determine the susceptibility by growing colonies from the first culture for another 24 hours. Here, the main goal is to examine the potential of infrared microscopy combined with multivariate analysis, to reduce the time it takes to identify Escherichia coli susceptibility to antibiotics and to determine the optimum choice of antibiotic to which the bacteria will respond. E coli colonies of the first culture from patients with urinary tract infections (UTI) were examined for the bacterial susceptibility using Fourier‐transform infrared (FTIR). Our results show that it is possible to determine the optimum choice of antibiotic with better than 89% sensitivity, in the time span of few minutes, following the first culture.      

Factors affecting the entry of antibiotics into Escherichia coli

Factors affecting the entry into Escherichia coli of diverse antibacterial agents, especially beta-lactams were investigated. Agents of greater than a critical molecular weight (approximately 600 Daltons) penetrated extremely poorly. However, there was little correlation between penetrative ability and molecular weight for substances below the critical size. Within classes of related antibiotics (e.g. cephalosporins) penetrative ability was highly dependent on hydrophobicity. The relationship was parabolic rather than linear in nature. The proposal that the envelope of E. coli preferentially excludes hydrophobic molecules is to some extent an artefact arising from pre-selection of the agents used. For unrelated antibiotics hydrophobic nature was a poor guide to penetrative ability. A rather empirical property, diffusion ability through agar, was found to show good inverse correlation with penetrative ability for many unrelated antibiotics.     

A transferable sucrose utilization approach for non-sucrose-utilizing Escherichia coli strains

Sucrose has economic and environmental advantages over glucose as a feedstock for bioprocesses. E. coli is widely used in industry, but the majority of current industrial E. coli strains cannot utilize sucrose. Previous attempts to transfer sucrose catabolic capabilities into non-sucrose-utilizing strains have met with limited success due to low growth rates on sucrose and phenotypic instability of the engineered strains. To address these problems, we developed a transferrable sucrose utilization cassette which confers efficient sucrose catabolism when integrated onto the E. coli chromosome. The cassette was based on the csc genes from E. coli W, a strain which grows very quickly on sucrose. Both plasmid-borne expression and chromosomal integration of a repressor-less sucrose utilizing cassette were investigated in E. coli strains K-12, B and C. In contrast to previous studies, strains harboring chromosomal cassettes could grow at the same rate as they do on glucose. Interestingly, we also discovered that spontaneous chromosomal integration of the csc genes was required to allow efficient growth from plasmid-transformed strains. The ability to engineer industrial strains for efficient sucrose utilization will allow substitution of sucrose for glucose in industrial fermentations. This will encourage the use of sucrose as a carbon source and assist in transition of our petrochemical-based economy to a bio-based economy.     

致病性大肠埃希菌血清型分布及对抗生素的敏感性分析

目的了解临床病例中致病性大肠埃希菌的主要血清型和对抗生素的敏感性。方法致病性大肠埃希菌的鉴定使用血清学的方法,药敏试验采用纸片扩散法,WHONET 5.0软件分析药敏结果。结果致病性大肠埃希菌的检出率为5.93%,共分离到7种血清型。在分离到的菌株中,ESBLs的检出率达45%。结论致病性大肠埃希菌是引起小儿腹泻的一种重要致病菌,应开展对致病性大肠埃希菌的检测,根据药敏结果选用合适药物。