Encapsulated <Emphasis Type="Italic">Escherichia coli</Emphasis> in alginate beads capable of secreting a heterologous pectin lyase

Background  

Production of heterologous proteins in the E. coli periplasm, or into the extracellular fluid has many advantages; therefore naturally occurring signal peptides are selected for proteins translocation. The aim of this study was the production in high yields of a recombinant pectin lyase that is efficiently secreted and the encapsulation of transformed E. coli cells for pectin degradation in a biotechnological process.     

Fate of Escherichia coli O145 present naturally in bovine slurry applied to vegetables before harvest,after washing and simulated wholesale and retail distribution

Aims

To determine the fate of Escherichia coli on vegetables that were processed through commercial wash treatments and stored under simulated retail conditions at 4°C or wholesale at fluctuating ambient temperatures (0–25°C, dependent on season).

Methods and Results

Bovine slurry that was naturally contaminated with E. coli O145 was applied without dilution or diluted 1:10 using borehole water to growing potatoes, leeks or carrots. Manure was applied 1 week prior to harvest to simulate a near‐harvest contamination event by manure deposition or an application of contaminated water to simulate a flooding event or irrigation from a contaminated water source. At harvest, crops were contaminated at up to 2 log cfu g^?1. Washing transferred E. coli into the water of a flotation tank used for potato washing and did not completely remove all traces of contamination from the crop. Manure‐contaminated potatoes were observed to contain 0·72 cfu E. coli O145 g^?1 after processing and retail storage. Manure‐contaminated leeks harboured 0·73–1·55 cfu E. coli O145 g^?1 after washing and storage. There was no cross‐contamination when leeks were spray washed. Washing in an abrasive drum resulted in less than perfect decontamination for manure‐contaminated carrots. There were five post‐distribution isolations from carrots irrigated with contaminated water 24 h prior to harvest.

Conclusions

Standard commercial washing and distribution conditions may be insufficient to reliably control human pathogenic E. coli on fresh produce.

Significance and Impact

Previous speculation that the cause of a UK foodborne disease outbreak was soil from imperfectly cleaned vegetables is plausible.     

Potential biological control of Erwinia tracheiphila by internal alimentary canal interactions in Acalymma vittatum with Pseudomonas fluorescens

Aims

We aim to determine if Pseudomonas fluorescens is a viable biological control for Erwinia tracheiphila within the insect vector, Acalymma vittatum.

Methods and Results

Pseudomonas fluorescens secreted fluorescein and inhibited growth of E. tracheiphila in disc diffusion assays. To determine if this antagonism was conserved within the insect vector, we performed in vivo assays by orally injecting beetles with bacterial treatments and fluorescent in situ hybridization to determine bacterial presence within the alimentary canal.

Conclusions

Pseudomonas fluorescens inhibited the growth of E. tracheiphila on a nutrient‐limiting medium. In situ experiments demonstrated that P. fluorescens is maintained within the alimentary canal of the beetle for at least 4 days, and co‐occurred with E. tracheiphila. When beetles were first presented with Pseudomonas and then challenged with E. tracheiphila, E. tracheiphila was not recovered via FISH after 4 days. These data suggest that P. fluorescens has potential as a biological control agent to limit E. tracheiphila within the insect vector.

Significance and Impact of the Study

This is a novel approach for controlling E. tracheiphila that has the potential to decrease reliance on insecticides, providing a safer environment for pollinators and growers.     

A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>, <Emphasis Type="Italic">E. moshkovskii</Emphasis> and <Emphasis Type="Italic">E. dispar</Emphasis> DNA in stool samples

Background  

E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples.     

Synergy between type 1 fimbriae expression and C3 opsonisation increases internalisation of E. coli by human tubular epithelial cells

Background  

Bacterial infection of the urinary tract is a common clinical problem with E. coli being the most common urinary pathogen. Bacterial uptake into epithelial cells is increasingly recognised as an important feature of infection. Bacterial virulence factors, especially fimbrial adhesins, have been conclusively shown to promote host cell invasion. Our recent study reported that C3 opsonisation markedly increases the ability of E. coli strain J96 to internalise into human proximal tubular epithelial cells via CD46, a complement regulatory protein expressed on host cell membrane. In this study, we further assessed whether C3-dependent internalisation by human tubular epithelial cells is a general feature of uropathogenic E. coli and investigated features of the bacterial phenotype that may account for any heterogeneity.     

Folding machineries displayed on a cation-exchanger for the concerted refolding of cysteine- or proline-rich proteins

Background  

Escherichia coli has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in E. coli frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant E. coli. Here, we describe the successful use of the immobilized folding machineries for in vitro refolding with the examples of high yield refolding of a ribonuclease A (RNase A) and cyclohexanone monooxygenase (CHMO).     

Secretion of flagellin by the LEE-encoded type III secretion system of enteropathogenic Escherichia coli

Background  

Enteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) pathogen that possesses a type III secretion system (T3SS) encoded within the locus of enterocyte effacement (LEE). The LEE is essential for A/E lesion formation and directs the secretion and translocation of multiple LEE-encoded and non-LEE encoded effector proteins into the cytosol of infected cells. In this study we used proteomics to compare proteins exported to the culture supernatant by wild type EPEC E2348/69, a ΔespADB mutant and a ΔescF T3SS mutant.     

Indole is an inter-species biofilm signal mediated by SdiA

Background  

As a stationary phase signal, indole is secreted in large quantities into rich medium by Escherichia coli and has been shown to control several genes (e.g., astD, tnaB, gabT), multi-drug exporters, and the pathogenicity island of E. coli; however, its impact on biofilm formation has not been well-studied.     

Functional analysis of the copy 1 of the fixNOQP operon of Ensifer meliloti under free‐living micro‐oxic and symbiotic conditions

Aim

In this work, phenotypic analyses of a Ensifer meliloti fixN1 mutant under free‐living and symbiotic conditions have been carried out.

Methods and Results

Ensifer meliloti fixN1 mutant showed a defect in growth as well as in TMPD‐dependent oxidase activity when cells were incubated under micro‐oxic conditions. Furthermore, haem c staining analyses of a fixN1 and a fixP1 mutant identified two membrane‐bound c‐type cytochromes of 27 and 32 kDa, present in microaerobically grown cells and in bacteroids, as the FixO and FixP components of the E. meliloti cbb₃ oxidase. Under symbiotic conditions, fixN1 mutant showed a clear nitrogen fixation defect in alfalfa plants that were grown in an N‐free nutrient solution during 3 weeks. However, in plants grown for a longer period, fixNOQP1 copy was not indispensable for symbiotic nitrogen fixation.

Conclusions

The copy 1 of the fixNOQP operon is involved in E. meliloti respiration and growth under micro‐oxic conditions as well as in the expression of the FixO and FixP components of the cbb₃ oxidase present in free‐living microaerobic cultures and in bacteroids. This copy is important for nitrogen fixation during the early steps of the symbiosis.

Significance and Impact of the Study

It is the first time that a functional analysis of the E. meliloti copy 1 of the fixNOQP operon is performed. In this work, the cytochromes c that constitute the cbb₃ oxidase operating in free‐living micro‐oxic cultures and in bacteroids of E. meliloti have been identified.     

Herd prevalence of Enterobacteriaceae producing CTX‐M‐type and CMY‐2 β‐lactamases among Japanese dairy farms

Aims

To determine the herd prevalence of Enterobacteriaceae producing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) among 381 dairy farms in Japan.

Methods and Results

Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 μg ml^?1). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX‐M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX‐M‐15 (n = 7), CTX‐M‐2 (n = 12), CTX‐M‐14 (n = 3), CMY‐2 (n = 2) or CTX‐M‐15/2/14 and CMY‐2 (n = 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (n = 23) and three genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed‐field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX‐M‐15 or CTX‐M‐27 was not detected.

Conclusions

Three clusters of CTX‐M (CTX‐M‐15, CTX‐M‐2, CTX‐M‐14) had spread among Japanese dairy farms.

Significance and Impact of the Study

This is the first report on the prevalence of multidrug‐resistant CTX‐M‐15–producing E. coli among Japanese dairy farms.     

The commonly-used DNA probe for diffusely-adherent Escherichia coli cross-reacts with a subset of enteroaggregative E. coli

Background  

The roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed.     

Properties and identification of antibiotic drug targets

Background  

We analysed 48 non-redundant antibiotic target proteins from all bacteria, 22 antibiotic target proteins from E. coli only and 4243 non-drug targets from E. coli to identify differences in their properties and to predict new potential drug targets.     

Inhibitory effects of Eucalyptus globulus on understorey plant growth and species richness are greater in non‐native regions

Aim

We studied the novel weapons hypothesis in the context of the broadly distributed tree species Eucalyptus globulus. We evaluated the hypothesis that this Australian species would produce stronger inhibitory effects on species from its non‐native range than on species from its native range.

Location

We worked in four countries where this species is exotic (U.S.A., Chile, India, Portugal) and one country where it is native (Australia).

Time period

2009–2012.

Major taxa studied

Plants.

Methods

We compared species composition, richness and height of plant communities in 20 paired plots underneath E. globulus individuals and open areas in two sites within its native range and each non‐native region. We also compared effects of litter leachates of E. globulus on root growth of seedlings in species from Australia, Chile, the U.S.A. and India.

Results

In all sites and countries, the plant community under E. globulus canopies had lower species richness than did the plant community in open areas. However, the reduction was much greater in the non‐native ranges: species richness declined by an average of 51% in the eight non‐native sites versus 8% in the two native Australian sites. The root growth of 15 out of 21 species from the non‐native range were highly suppressed by E. globulus litter leachates, whereas the effect of litter leachate varied from facilitation to suppression for six species native to Australia. The mean reduction in root growth for Australian plants was significantly lower than for plants from the U.S.A., Chile and India.

Main conclusions

Our results show biogeographical differences in the impact of an exotic species on understorey plant communities. Consistent with the novel weapons hypothesis, our findings suggest that different adaptations of species from the native and non‐native ranges to biochemical compounds produced by an exotic species may play a role in these biogeographical differences.     

Two distinct regions in the model protein Peb1 are critical for its heterologous transport out of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli is frequently the first-choice host organism in expression of heterologous recombinant proteins in basic research as well as in production of commercial, therapeutic polypeptides. Especially the secretion of proteins into the culture medium of E. coli is advantageous compared to intracellular production due to the ease in recovery of the recombinant protein. Since E. coli naturally is a poor secretor of proteins, a few strategies for optimization of extracellular secretion have been described. We have previously reported efficient secretion of the diagnostically interesting model protein Peb1 of Campylobacter jejuni into the growth medium of Escherichia coli strain MKS12 (ΔfliCfliD). To generate a more detailed understanding of the molecular mechanisms behind this interesting heterologous secretion system with biotechnological implications, we here analyzed further the transport of Peb1 in the E. coli host.     

Evaluation of Petrifilm™ Select <Emphasis Type="Italic">E. coli</Emphasis> Count Plate medium to discriminate antimicrobial resistant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Screening and enumeration of antimicrobial resistant Escherichia coli directly from samples is needed to identify emerging resistant clones and obtain quantitative data for risk assessment. Aim of this study was to evaluate the performance of 3M™ Petrifilm™ Select E. coli Count Plate (SEC plate) supplemented with antimicrobials to discriminate antimicrobial-resistant and non-resistant E. coli.     

Proteomics of Porphyromonas gingivalis within a model oral microbial community

Background  

Porphyromonas gingivalis is a periodontal pathogen that resides in a complex multispecies microbial biofilm community known as dental plaque. Confocal laser scanning microscopy showed that P. gingivalis can assemble into communities in vitro with Streptococcus gordonii and Fusobacterium nucleatum, common constituents of dental plaque. Whole cell quantitative proteomics, along with mutant construction and analysis, were conducted to investigate how P. gingivalis adapts to this three species community.     

Herd-level risk factors associated with the presence of Phage type 21/28 E. coli O157 on Scottish cattle farms

Background  

E. coli O157 is a bacterial pathogen that is shed by cattle and can cause severe disease in humans. Phage type (PT) 21/28 is a subtype of E. coli O157 that is found across Scotland and is associated with particularly severe human morbidity.     

Role of the ArcAB two-component system in the resistance of Escherichia coli to reactive oxygen stress

Background  

The global regulatory system ArcAB controls the anaerobic growth of E. coli, however, its role in aerobic conditions is not well characterized. We have previously reported that ArcA was necessary for Salmonella to resist reactive oxygen species (ROS) in aerobic conditions.