Reconstitution of the GalP galactose transport activity of Escherichia coli into liposomes made from soybean phospholipids

Galactose transport activity from Escherichia coli was solubilized with octyl glucoside, and reconstituted into liposomes made from soybean or E. coli lipid. Galactose counterflow in the proteoliposomes was inhibited by glucose, talose, 2-deoxygalactose and 6-deoxygalactose, confirming that it was due to GalP and not one of the other E. coli galactose transport systems.     

The role of the catalytic domain of E. coli GluRS in tRNA discrimination

Discrimination of tRNA^Gln is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNA^Gln and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNA^Gln. Our results demonstrate that in addition to the anticodon-binding domain, tRNA^Gln discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.     

Prevalence of Escherichia coli strains resistance to antibiotics in wound infections and raw milk

Antibiotic-resistant Escherichia coli strains including extended-spectrum β-lactamase (ESBL) isolates are globally widespread in medical, food, and environmental sources. Some of these strains are considered the most pathogenic bacteria in humans. The present work examined the predominance of antibiotic resistance in E. coli strains in wound infections comparing with E. coli strains isolated from a raw milk as a potential source of those strains. The wound infections included abdomen, anus, arm, back, buttock, chest, foot, hand, head, leg, lung, mouth, neck, penis, thigh, toe, and vagina infections. In total, 161 and 153 isolates identified as E. coli were obtained from wound infections and raw milk, respectively. A Vitek 2 system innovated by bioMérieux, France was applied to perform the identification and susceptibility tests. The E. coli isolates that have ability to produce ESBL were detected by an ESBL panel and NO45 card (bioMérieux). Over half of the E. coli were from abdomen, back, and buttock wound infections. More than 50%of the E. coli isolates obtained from wound infections were resistant to cefazolin, ampicillin, cefuroxime, ciprofloxacin, mezlocillin, moxifloxacin, piperacillin, and tetracycline; 70% of the isolates from wound infections and 0% of the isolates from raw milk were E. coli isolates produced ESBL. The data showed that the strains resistance to multi-antibiotic and produced ESBL are more widespread among wound infections than in raw milk.     

Survival of Escherichia coli in the environment: fundamental and public health aspects

In this review, our current understanding of the species Escherichia coli and its persistence in the open environment is examined. E. coli consists of six different subgroups, which are separable by genomic analyses. Strains within each subgroup occupy various ecological niches, and can be broadly characterized by either commensalistic or different pathogenic behaviour. In relevant cases, genomic islands can be pinpointed that underpin the behaviour. Thus, genomic islands of, on the one hand, broad environmental significance, and, on the other hand, virulence, are highlighted in the context of E. coli survival in its niches. A focus is further placed on experimental studies on the survival of the different types of E. coli in soil, manure and water. Overall, the data suggest that E. coli can persist, for varying periods of time, in such terrestrial and aquatic habitats. In particular, the considerable persistence of the pathogenic E. coli O157:H7 is of importance, as its acid tolerance may be expected to confer a fitness asset in the more acidic environments. In this context, the extent to which E. coli interacts with its human/animal host and the organism''s survivability in natural environments are compared. In addition, the effect of the diversity and community structure of the indigenous microbiota on the fate of invading E. coli populations in the open environment is discussed. Such a relationship is of importance to our knowledge of both public and environmental health.     

Flagella proteins contribute to the production of outer membrane vesicles from Escherichia coli W3110

Gram-negative bacteria, including Escherichia coli, release outer membrane vesicles (OMVs) that are derived from the bacterial outer membrane. OMVs contribute to bacterial cell–cell communications and host–microbe interactions by delivering components to locations outside the bacterial cell. In order to explore the molecular machinery involved in OMV biogenesis, the role of a major OMV protein was examined in the production of OMVs from E. coli W3110, which is a widely used standard E. coli K-12 strain. In addition to OmpC and OmpA, which are used as marker proteins for OMVs, an analysis of E. coli W3110 OMVs revealed that they also contain abundant levels of FliC, which is also known as flagellin. A membrane-impermeable biotin-labeling reagent did not label FliC in intact OMVs, but labeled FliC in sonically disrupted OMVs, suggesting that FliC is localized in the lumen of OMV. Compared to the parental strain expressing wild-type fliC, an E. coli strain with a fliC-null mutation produced reduced amounts of OMVs based on both protein and phosphate levels. In addition, an E. coli W3110-derived strain with a null-mutation in flgK, which encodes flagellar hook-associated protein that is essential along with FliC for flagella synthesis, also produced fewer OMVs than the parental strain. Taken together, these results indicate that the ability to form flagella, including the synthesis of flagella proteins, affects the production of E. coli W3110 OMVs.     

Occurrence of Plasmid-Mediated AmpC β-Lactamases Among Escherichia coli and Klebsiella pneumoniae Clinical Isolates in a Tertiary Care Hospital in Bangalore

Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC β-lactamases are limited because these organisms are usually resistant to all the β-lactam antibiotics, except for cefepime, cefpirome and the carbapenems. These organisms are a major concern in nosocomial infections and should therefore be monitored in surveillance studies. Hence, this study was aimed out to determine the prevalence of plasmid-mediated AmpC β-lactamases in E. coli and K. pneumoniae from a tertiary care in Bangalore. A total of 63 E. coli and 27 K. pneumoniae were collected from a tertiary care hospital in Bangalore from February 2008 to July 2008. The isolates with decreased susceptibility to cefoxitin were subjected to confirmation test with three dimensional extract tests. Minimum inhibitory concentrations (MICs) were determined by agar dilution method. Conjugation experiments, plasmid profiling and susceptibility testing were carried out to investigate the underlying mechanism of resistance. In our study, 52 (57.7%) isolates showed resistance to cefoxitin, the occurrence of AmpC was found to be 7.7% of the total isolates. Plasmid analysis of the selected isolates showed the presence of a single plasmid of 26 kb in E. coli and 2 Kb in K. pneumoniae. Plasmid-mediated AmpC β-lactamases were found in 11.1% of K. pneumoniae and in 6.3% of E. coli. Curing and conjugation experiments showed that resistance to cephamycins and cephalosporins was plasmid-mediated. Our study has demonstrated the occurrence of plasmid-mediated AmpC in E. coli and K. pneumoniae which illustrates the importance of molecular surveillance in tracking AmpC-producing strains at general hospitals and emphasizes the need for epidemiological monitoring.     

Potentiation of antibiotic activity by Passiflora cincinnata Mast. front of strains Staphylococcus aureus and Escherichia coli

The development of new drugs from plants is an interesting alternative approach to overcoming microbial resistance. Passiflora cincinnata shows resistance to diseases and pests and a higher concentration of chemical components that may be useful in the pharmaceutical industry. We investigated the potential antimicrobial and antibiotic-modifying activity of hydroalcoholic extracts of leaves, stems, bark, pulp and seeds of P. cincinnata. The extracts were prepared by homogenization of material in 50% ethanol. Minimum inhibitory concentration (MIC) was determined by the broth dilution method, and the bacterial strains tested were Staphylococcus aureus and Escherichia coli. Antibiotic-modifying activity was evaluated against the strains S. aureus 03 and E. coli 08, using a subinhibitory concentration of extract. The antibiotics tested were: amikacin, gentamicin, ampicillin, potassium benzylpenicillin and oxacillin. The extracts did not show antimicrobial activity of clinical relevance, where the MIC was equal to or greater than 1024 μg/mL. S. aureus showed 13 events, while E. coli showed only 4 events. Among these events, 14 involved synergistic activity, potentiating the effect of the antibiotics, and only 3 events demonstrated antagonistic activity toward ampicillin. Hydroalcoholic extracts are potential antimicrobial agents when combined with conventional drugs little utilized in in vivo treatment.     

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.     

Molecular cloning of a maltose transport gene from Bacillus stearothermophilus and its expression in Escherichia coli K-12

Genes responsible for maltose utilization from Bacillus stearothermophilus ATCC7953 were cloned in the plasmid vector pBR325 and functionally expressed in Escherichia coli. The 4.2 kb Bacillus DNA insert in clone pAM1750 suppressed the growth defects on maltose caused by mutations in E. coli maltose transport genes (malE, malK or complete malB deletion) but not mutations in genes affecting intracellular maltose metabolism (malA region). Transport studies in E. coli and B. stearothermophilus suggested that pAM1750 codes for a high affinity transport system, probably one of two maltose uptake systems found in B. stearothermophilus ATCC7953. Nucleotide sequence analysis of a 3.6 kb fragment of pAM 1750 revealed three open reading frames (ORFs). One of the ORFs, malA, encoded a putative hydrophobic protein with 12 potential transmembrane segments. MalA showed amino acid sequence similarity to proteins in the superfamily containing LacY lactose permease and also some similarity to MaIG protein, a member of a binding protein-dependent transport system in E. coli. The products of two other ORFs were not hydrophobic, did not show similarity to other known sequences and were found not to be essential for maltose utilization in transport-defective E. coli mutants. Hence MalA protein was the only protein necessary for maltose transport, but despite giving a detectable but low level of transport function in E. coli, the protein was very poorly expressed and could not be identified.     

Metabolic Engineering for Resveratrol Derivative Biosynthesis in Escherichia coli

We previously reported that the SbROMT3syn recombinant protein catalyzes the production of the methylated resveratrol derivatives pinostilbene and pterostilbene by methylating substrate resveratrol in recombinant E. coli. To further study the production of stilbene compounds in E. coli by the expression of enzymes involved in stilbene biosynthesis, we isolated three stilbene synthase (STS) genes from rhubarb, peanut, and grape as well as two resveratrol O-methyltransferase (ROMT) genes from grape and sorghum. The ability of RpSTS to produce resveratrol in recombinant E. coli was compared with other AhSTS and VrSTS genes. Out of three STS, only AhSTS was able to produce resveratrol from p-coumaric acid. Thus, to improve the solubility of RpSTS, VrROMT, and SbROMT3 in E. coli, we synthesized the RpSTS, VrROMT and SbROMT3 genes following codon-optimization and expressed one or both genes together with the cinnamate/4-coumarate:coenzyme A ligase (CCL) gene from Streptomyces coelicolor. Our HPLC and LC-MS analyses showed that recombinant E. coli expressing both ScCCL and RpSTSsyn led to the production of resveratrol when p-coumaric acid was used as the precursor. In addition, incorporation of SbROMT3syn in recombinant E. coli cells produced resveratrol and its mono-methylated derivative, pinostilbene, as the major products from p-coumaric acid. However, very small amounts of pterostilbene were only detectable in the recombinant E. coli cells expressing the ScCCL, RpSTSsyn and SbROMT3syn genes. These results suggest that RpSTSsyn exhibits an enhanced enzyme activity to produce resveratrol and SbROMT3syn catalyzes the methylation of resveratrol to produce pinostilbene in E. coli cells.     

Effect of Proximity to a Cattle Feedlot on Escherichia coli O157:H7 Contamination of Leafy Greens and Evaluation of the Potential for Airborne Transmission

The impact of proximity to a beef cattle feedlot on Escherichia coli O157:H7 contamination of leafy greens was examined. In each of 2 years, leafy greens were planted in nine plots located 60, 120, and 180 m from a cattle feedlot (3 plots at each distance). Leafy greens (270) and feedlot manure samples (100) were collected six different times from June to September in each year. Both E. coli O157:H7 and total E. coli bacteria were recovered from leafy greens at all plot distances. E. coli O157:H7 was recovered from 3.5% of leafy green samples per plot at 60 m, which was higher (P < 0.05) than the 1.8% of positive samples per plot at 180 m, indicating a decrease in contamination as distance from the feedlot was increased. Although E. coli O157:H7 was not recovered from air samples at any distance, total E. coli was recovered from air samples at the feedlot edge and all plot distances, indicating that airborne transport of the pathogen can occur. Results suggest that risk for airborne transport of E. coli O157:H7 from cattle production is increased when cattle pen surfaces are very dry and when this situation is combined with cattle management or cattle behaviors that generate airborne dust. Current leafy green field distance guidelines of 120 m (400 feet) may not be adequate to limit the transmission of E. coli O157:H7 to produce crops planted near concentrated animal feeding operations. Additional research is needed to determine safe set-back distances between cattle feedlots and crop production that will reduce fresh produce contamination.     

Effect of Fe/Al Hydroxides on Transport and Retention of Escherichia coli in Saturated Sand Media

The transport of bacteria has been investigated extensively using iron (Fe) (hydr)oxide-coated quartz. However, few studies have investigated the effects of aluminum (Al) (hydr)oxide on the transport of bacteria. In this study, column experiments were conducted to investigate the effects of Fe/Al hydroxides on the transport of Escherichia coli (E. coli) in saturated quartz sand at different pH levels, ionic strengths (IS), and ionic compositions. Fe/Al hydroxide coatings increased the positive charge of quartz, reduced the negative charge, shifted zeta potential in a positive direction, and thus enhanced the retention of E. coli on quartz. The retention of E. coli decreased with increasing pH and increased with increasing IS. These findings were consistent with the theoretical prediction of the Derjaguin, Landau, Verwey and Overbeek (DLVO) interaction energy. Calcium ions improved the retention of E. coli in the column. Since Al-hydroxide-coated quartz had a more positive charge, the retention of E. coli was higher in Al-hydroxide-coated quartz than in Fe-hydroxide-coated quartz. When compared with quartz alone, Fe/Al hydroxide coatings significantly reduced the transport of E. coli, and the inhibitory effect of Al hydroxide was greater than that of Fe hydroxide.     

Electrostatic interactions of colicin E1 with the surface of Escherichia coli total lipid

The surface properties of colicin E1, a 522-amino acid protein, and its interaction with monolayers of Escherichia coli (E. coli) total lipid and 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DOPC) were studied using the Langmuir-Blodgett (LB) technique. Colicin E1 is amphiphilic, forming a protein monolayer at the air/buffer interface. The protein is thought to interact with the E. coli total lipid head groups through electrostatic interactions, followed by its insertion into the lipid monolayers. Supported lipid bilayers (SLBs) of E. coli total lipid and DOPC, deposited onto mica at the cell membrane equivalence pressure for E. coli and incubated with colicin E1, were imaged by contact mode atomic force microscopy (CM-AFM). Colicin E1 formed protein aggregates on DOPC SLBs, while E. coli total lipid SLB was deformed following its incubation with colicin E1. Corresponding lateral force images, along with electrostatic surface potentials for colicin E1 P190, imply a direct interaction of colicin E1 with lipid head groups facilitating their charge neutralization.     

Interaction of Escherichia coli and Soil Particles in Runoff

A laboratory-scale model system was developed to investigate the transport mechanisms involved in the horizontal movement of bacteria in overland flow across saturated soils. A suspension of Escherichia coli and bromide tracer was added to the model system, and the bromide concentration and number of attached and unattached E. coli cells in the overland flow were measured over time. Analysis of the breakthrough curves indicated that the E. coli and bromide were transported together, presumably by the same mechanism. This implied that the E. coli was transported by advection with the flowing water. Overland-flow transport of E. coli could be significantly reduced if the cells were preattached to large soil particles (>45 μm). However, when unattached cells were inoculated into the system, the E. coli appeared to attach predominantly to small particles (<2 μm) and hence remained unattenuated during transport. These results imply that in runoff generated by saturation-excess conditions, bacteria are rapidly transported across the surface and have little opportunity to interact with the soil matrix.     

A complete view of the genetic diversity of the Escherichia coli O-antigen biosynthesis gene cluster

The O antigen constitutes the outermost part of the lipopolysaccharide layer in Gram-negative bacteria. The chemical composition and structure of the O antigen show high levels of variation even within a single species revealing itself as serological diversity. Here, we present a complete sequence set for the O-antigen biosynthesis gene clusters (O-AGCs) from all 184 recognized Escherichia coli O serogroups. By comparing these sequences, we identified 161 well-defined O-AGCs. Based on the wzx/wzy or wzm/wzt gene sequences, in addition to 145 singletons, 37 serogroups were placed into 16 groups. Furthermore, phylogenetic analysis of all the E. coli O-serogroup reference strains revealed that the nearly one-quarter of the 184 serogroups were found in the ST10 lineage, which may have a unique genetic background allowing a more successful exchange of O-AGCs. Our data provide a complete view of the genetic diversity of O-AGCs in E. coli showing a stronger association between host phylogenetic lineage and O-serogroup diversification than previously recognized. These data will be a valuable basis for developing a systematic molecular O-typing scheme that will allow traditional typing approaches to be linked to genomic exploration of E. coli diversity.     

High-throughput Sorting of an Anticalin Library via EspP-mediated Functional Display on the Escherichia coli Cell Surface

We demonstrate that small engineered single-chain binding proteins based on the lipocalin scaffold, so-called Anticalins, can be functionally displayed on the Gram-negative bacterial cell envelope. To this end, the β-domains of five different bacterial autotransporters (the IgA protease from Neisseria gonorrhoeae, the esterase EstA from Pseudomonas aeruginosa, the YpjA autotransporter from E. coli K12, the AIDA-I adhesin from enteropathogenic E. coli O127:H27 strain 2787 and the protease EspP from enterohemorrhagic E. coli O157:H7 strain EDL933) were compared with respect to display level, functional variance, and bacterial cell viability. Use of the EspP autotransporter led to a system with high genetic stability for the display of fully functional Anticalins in high density on the cell surface of E. coli as shown by quantitative flow cytofluorimetry. This system was applied to engineer an immunostimulatory Anticalin that binds and blocks the extracellular region of human CTLA-4 to achieve a slower dissociation rate. A combinatorial library of the original Anticalin was generated by error-prone PCR, subjected to E. coli cell surface display, and applied to repeated cycles of cell sorting after incubation with the fluorescently labelled target protein under competition with the unlabelled extracellular domain of CTLA-4. The resulting Anticalin variants, which were expressed and purified as soluble proteins, showed more than eightfold decelerated target dissociation, as revealed by real time surface plasmon resonance analysis. Hence, the EspP autotransporter-mediated E. coli surface display in combination with high-throughput fluorescence-activated cell sorting (FACS) provides an efficient strategy to select for Anticalins, and possibly other small protein scaffolds, with improved binding properties, which is particularly useful for in vitro affinity maturation but may also serve for the selection of novel target specificity from naive libraries.     

改良环介导等温扩增技术快速检测肉类中的大肠杆菌O157: H7

针对大肠杆菌O157:H7(Escherichia coli O157:H7,E.coli O157:H7)传统检测方法检测周期长的问题,建立了肉类中的E.coli O157:H7的改良环介导等温扩增(LAMP)快速检测方法。以E.coli O157:H7的O157特异性抗原rfbE基因、鞭毛H7特异性抗原fliC基因序列作为靶序列,分别设计2套增加了环引物的改良LAMP引物序列,单管同时检测,通过肉眼观察白色沉淀,判断检测结果。采用36株细菌验证了该改良LAMP引物的特异性。热裂解法提取的DNA经改良LAMP体系扩增20 min,检测E.coli O157:H7的灵敏度为1.4 CFU/mL,人工污染肉中的E.coli O157:H7检出限为1.8 CFU/g。137份实样中,检测出1份E.coli O157:H7假阳性,与行业标准SNT0973-2000符合率达到99.3%。     

Insights into the evolution of gene organization and multidrug resistance from Klebsiella pneumoniae plasmid pKF3-140

Plasmid-mediated transfer of drug-resistance genes among various bacterial species is considered one of the most important mechanisms for the spread of multidrug resistance. To gain insights into the evolution of gene organization and antimicrobial resistance in clinical bacterial samples, a complete plasmid genome of Klebsiella pneumoniae pKF3-140 is determined, which has a circular chromosome of 147,416 bp in length. Among the 203 predicted genes, 142 have function assignment and about 50 appear to be involved in plasmid replication, maintenance, conjugative transfer, iron acquisition and transport, and drug resistance. Extensive comparative genomic analyses revealed that pKF3-140 exhibits a rather low sequence similarity and structural conservation with other reported K. pneumoniae plasmids. In contrast, the overall organization of pKF3-140 is highly similar to Escherichia coli plasmids p1ESCUM and pUTI89, which indicates the possibility that K. pneumoniae pKF3-140 may have a potential origin in E. coli. Meanwhile, interestingly, several drug resistant genes show high similarity to the plasmid pU302L in Salmonella enterica serovar Typhimurium U302 strain G8430 and the plasmid pK245 in K. pneumoniae. This mosaic pattern of sequence similarities suggests that pKF3-140 might have arisen from E. coli and acquired the resistance genes from a variety of enteric bacteria and underscores the importance of a further understanding of horizontal gene transfer among enteric bacteria.