Influence of specific growth rate on specific productivity and glycosylation of a recombinant avidin produced by a Pichia pastoris Mut+ strain

A recombinant avidin-producing Mut+ Pichia pastoris strain was used as a model organism to study the influence of the methanol feeding strategy on the specific product productivity (q(p)) and protein glycosylation. Fed-batch cultivations performed at various specific growth rates (micro) and residual methanol concentrations showed that the specific avidin productivity is growth-dependent. The specific productivity increases strongly with the specific growth rate for micro ranging from 0 to 0.02 h(-1), and increases only slightly with the specific growth rate above this limit. N-terminal glycosylation was also found to be influenced by the specific growth rate, since 9-mannose glycans were the most abundant form at low growth rates, whereas 10-mannose carbohydrate chains were favored at higher micro. These results show that culture parameters, such as the specific growth rate, may significantly affect the activity of glycoproteins produced in Pichia pastoris. In terms of process optimization, this suggests that a compromise on the specific growth rate may have to be found, in certain cases, to work with an acceptable productivity while avoiding the addition of many mannoses.     

Reduction of Escherichia coli O157:H7 on radish seeds by sequential application of aqueous chlorine dioxide and dry‐heat treatment

Aims: To assess the effectiveness of sequential treatments of radish seeds with aqueous chlorine dioxide (ClO₂) and dry heat in reducing the number of Escherichia coli O157:H7. Methods and Results: Radish seeds containing E. coli O157:H7 at 5·5 log CFU g^?1 were treated with 500 μg ml^?1 ClO₂ for 5 min and subsequently heated at 60°C and 23% relative humidity for up to 48 h. Escherichia coli O157:H7 decreased by more than 4·8 log CFU g^?1 after 12 h dry‐heat treatment. The pathogen was inactivated after 48 h dry‐heat treatment, but the germination rate of treated seeds was substantially reduced from 91·2 ± 5·0% to 68·7 ± 12·3%. Conclusions: Escherichia coli O157:H7 on radish seeds can be effectively reduced by sequential treatments with ClO₂ and dry heat. To eliminate E. coli O157:H7 on radish seeds without decreasing the germination rate, partial drying of seeds at ambient temperature before dry‐heat treatment should be investigated, and conditions for drying and dry‐heat treatment should be optimized. Significance and Impact of the study: This study showed that sequential treatment with ClO₂ and dry‐heat was effective in inactivating large numbers of E. coli O157:H7 on radish seeds. These findings will be useful when developing sanitizing strategies for seeds without compromising germination rates.     

Improved PCR assay for the specific detection and quantitation of Escherichia coli serotype O157 in water

Escherichia coli serotype O157 is still a major global healthcare problem. However, only limited information is now available on the molecular and serological detection of pathogenic bacteria. Therefore, the development of appropriate strategies for their rapid identification and monitoring is still needed. In general, the sequence analysis based on stx, slt, eae, hlyA, rfb, and fliC _h7 genes is widely employed for the identification of E. coli serotype O157; but there have been critical defects in the diagnosis and identification of E. coli serotype O157, in that they are also present in other E. coli serogroups. In this study, NCBI-BLAST searches using the nucleotide sequences of the putative regulatory protein gene from E. coli O157:H7 str. Sakai found sequence difference at the serotype level. The specific primers from the putative regulatory protein gene were designed and investigated for their sensitivity and specificity for detecting the pathogen in environment water samples. The specificity of the primer set was evaluated using genomic DNA from 8 isolates of E. coli serotype O157 and 32 other reference strains. In addition, the sensitivity and specificity of this assay were confirmed by successful identification of E. coli serotype O157 in environmental water samples. In conclusion, this study showed that the newly developed quantitative serotype-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk E. coli serotype O157.     

Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness

With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X-press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (q_S) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy-chain single-domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X-press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and q_S are used to control productivity and leakiness. In the X-press strain, extracellular SpA and VHH titer reach up to 349 and 19.6 mg g⁻¹, respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X-press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.     

Is lack of susceptible recipients in the intestinal environment the limiting factor for transduction of Shiga toxin-encoding phages?

Aim: To determine whether a Shiga toxin 2 (Stx2)-encoding phage from Escherichia coli O157:H7 could be transmitted to commensal E. coli in a ruminant host without adding a specific recipient strain. Methods and Results: Sheep were inoculated with an E. coli O157:H7 strain containing an Stx2-encoding bacteriophage (Φ3538) in which a chloramphenicol-resistant gene, cat, is inserted into stx₂. A total of 149 faecal samples were sampled and analysed for detection and quantification of E. coli O157:H7 and presumptive transductants. Phage Φ3538 (Δstx₂::cat) was demonstrated to be transduced to an ovine E. coli O175:H16 at one occasion. Conclusions: The study demonstrates an in vivo transduction in sheep from an E. coli O157:H7 strain to an ovine E. coli O175:H16. A functional Stx2-encoding phage was incorporated into the host’s DNA. Significance and Impact of the Study: This is the first in vivo stx phage transduction study reported in which a recipient strain was not fed to the test animals. We suggest that the access to susceptible hosts is one main limiting factor for transduction to occur in the intestine.     

Development of a combined immunochromatographic lateral flow assay for accurate and rapid Escherichia coli O157:H7 detection

Escherichia coli O157:H7 is an important pathogenic Bacterium that threatens human health. A convenient, sensitive and specific method for the E. coli O157:H7 detection is necessary. We developed two pairs of monoclonal antibodies through traditional hybridoma technology, one specifically against E. coli O157 antigen and the other specifically against E. coli H7 antigen. Using these two pairs of antibodies, we developed two rapid test kits to specifically detect E. coli O157 antigen and E. coli H7 antigen, respectively. The detection sensitivity for O157 positive E. coli is 1 × 10³ CFU per ml and for H7 positive E. coli is 1 × 10⁴ CFU per ml. Combining these two pairs of antibodies together, we developed a combo test strip that can specifically detect O157: H7, with a detection sensitivity of 1 × 10⁴ CFU per ml, when two detection lines are visible to the naked eye. This is currently the only rapid detection reagent that specifically detects O157: H7 by simultaneously detecting O157 antigen and H7 antigens of E. coli. Our product has advantages of simplicity and precision, and can be a very useful on-site inspection tool for accurate and rapid detection of E. coli O157:H7 infection.     

Effect of curli expression and hydrophobicity of Escherichia coli O157:H7 on attachment to fresh produce surfaces

Aim: To investigate the effect of curli expression on cell hydrophobicity, biofilm formation and attachment to cut and intact fresh produce surfaces. Methods and Results: Five Escherichia coli O157:H7 strains were evaluated for curli expression, hydrophobicity, biofilm formation and attachment to intact and cut fresh produce (cabbage, iceberg lettuce and Romaine lettuce) leaves. Biofilm formation was stronger when E. coli O157:H7 were grown in diluted tryptic soy broth (1 : 10). In general, strong curli‐expressing E. coli O157:H7 strains 4406 and 4407 were more hydrophobic and attached to cabbage and iceberg lettuce surfaces at significantly higher numbers than other weak curli‐expressing strains. Overall, E. coli O157:H7 populations attached to cabbage and lettuce (iceberg and Romaine) surfaces were similar (P > 0·05), indicating produce surfaces did not affect (P < 0·05) bacterial attachment. All E. coli O157:H7 strains attached rapidly on intact and cut produce surfaces. Escherichia coli O157:H7 attached preferentially to cut surfaces of all produce types; however, the difference between E. coli O157:H7 populations attached to intact and cut surfaces was not significant (P > 0·05) in most cases. Escherichia coli O157:H7 attachment and attachment strength (S_R) to intact and cut produce surfaces increased with time. Conclusions: Curli‐producing E. coli O157:H7 strains attach at higher numbers to produce surfaces. Increased attachment of E. coli O157:H7 on cut surfaces emphasizes the need for an effective produce wash to kill E. coli O157:H7 on produce. Significance and Impact of the Study: Understanding the attachment mechanisms of E. coli O157:H7 to produce surfaces will aid in developing new intervention strategies to prevent produce outbreaks.     

Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha‐L‐iduronidase

The Brassica rapa hairy root based expression platform, a turnip hairy root based expression system, is able to produce human complex glycoproteins such as the alpha—L—iduronidase (IDUA) with an activity similar to the one produced by Chinese Hamster Ovary (CHO) cells. In this article, a particular attention has been paid to the N‐ and O‐glycosylation that characterize the alpha‐L‐iduronidase produced using this hairy root based system. This analysis showed that the recombinant protein is characterized by highly homogeneous post translational profiles enabling a strong batch to batch reproducibility. Indeed, on each of the 6 N‐glycosylation sites of the IDUA, a single N‐glycan composed of a core Man₃GlcNAc₂ carrying one beta(1,2)‐xylose and one alpha(1,3)‐fucose epitope (M3XFGN2) was identified, highlighting the high homogeneity of the production system. Hydroxylation of proline residues and arabinosylation were identified during O‐glycosylation analysis, still with a remarkable reproducibility. This platform is thus positioned as an effective and consistent expression system for the production of human complex therapeutic proteins.     

Escherichia coli O157 Somatic Antigen Is Present in an Isolate of E. fergusonii

A bacterium that tested positive with antibodies specific for Escherichia coli O157 was isolated from beef during routine screening procedures. The bacterium was identified as E. fergusonii by biochemical testing and partial sequencing of 16S rRNA. The isolate was tested for the presence of genes encoding Shiga toxins, the E. coli attaching and effacing factor, enterohemolysin, and the O157 O antigen. The isolate tested negative for Shiga toxins and other E. coli O157 virulence markers but was found to harbor the genes encoding the O157 antigen. These results suggest genetic transfer of the O antigen gene cluster between E. coli O157:H7 and E. fergusonii.     

Non-O157:H7 Shiga toxin (verocytotoxin)-producing Escherichia coli strains: epidemiological significance and microbiological diagnosis

Shiga toxin (Stx)-producing Escherichia coli (STEC) are important causes of diarrhoea and the haemolytic uremic syndrome (HUS). The most common STEC serotype implicated worldwide is E. coli O157:H7 that is diagnosed using procedures based on its typical phenotypic feature, the lack of sorbitol fermentation. In addition to E. coli O157:H7, a variety of non-O157:H7 STEC strains that usually ferment sorbitol and are thus missed by using the diagnostic protocol for E.coli O157:H7 have been isolated from patients. Among these sorbitol-fermenting (SF) non-O157:H7 STEC, SF E. coli O157:H⁻ and non-O157 STEC strains of serogroups O26, O103, O111 and O145 have emerged as significant causes of HUS and diarrhoea in continental Europe and have been associated with human disease in other parts of the world. Microbiological diagnosis of non-O157:H7 STEC strains is difficult due to their serotype diversity and the absence of a simple biochemical property that distinguishes such strains from the physiological intestinal microflora. Screening for non-O157:H7 STEC and their isolation from stools is presently based on the detection of Stx production or stx genes that are common characteristics of such strains. Molecular subtyping of the most frequent non-O157 STEC demonstrated that strains of serogroups O26, O103 and O111 belong to their own clonal lineages and show unique virulence profiles. SF STEC O157:H⁻ strains that have been isolated mostly in Central Europe represent a new clone within E. coli O157 serogroup which has its own typical combination of virulence factors. This revised version was published online in November 2006 with corrections to the Cover Date.     

Prevalence of verotoxigenic Escherichia coli O157 (VTEC O157) and compliance with microbiological safety standards in bovine carcasses from an industrial beef slaughter plant

The presence of Salmonella spp. and levels of Enterobacteriaceae and aerobic plate count were determined in 300 bovine carcasses randomly collected in an industrial cattle slaughterhouse in Catalonia (Spain) as part of a control programme to validate good slaughter practices according to Commission Regulation No 2073/2005. The verotoxigenic Escherichia coli O157 (VTEC O157), although not currently legislated, was also investigated in the same carcasses due to the importance of bovines as a reservoir for this micro‐organism. Virulence genes (vtx1, vtx2 and eae), the presence of fliC_H7 and antimicrobial susceptibility were studied in E. coli O157 isolates. Levels of Enterobacteriaceae and aerobic colonies and the presence of Salmonella were within the admissible range stipulated by current legislation. However, VTEC O157 was detected in 14·7% of carcasses. Among the VTEC O157 strains tested for antimicrobial susceptibility, 65% were multiresistant. Overall, the results of this study indicate that even with good manufacturing practices, contamination with VTEC O157 can occur and cattle meat can pose a risk to human health. These results confirm the need for a review of the appropriateness of introducing antimicrobial treatments in the processing of cattle carcasses in Europe.

Significance and Impact of the Study

This study describes the prevalence of verotoxigenic and multidrug‐resistant E. coli O157 strains in bovine carcasses. These results suggest that despite the good manufacturing practices used in the slaughterhouse studied (the largest in Catalonia slaughtering over 81 000 cattle per year), the absence of verotoxigenic E. coli O157 in bovine carcasses cannot be guaranteed.     

Productivity improvement of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation via robust optimization

A nonlinear model of a recombinant Escherichia coli producing porcine growth hormone (pGH) fermentation was developed. The model was used to calculate a glucose feeding and temperature strategy to optimize the production of pGH. Simulations showed that the implementation of optimal feed and temperature profiles was sensitive to the maximum specific growth rate, and a mismatch could result in excessive acetate production and a significant reduction in pGH yield. An optimization algorithm was thus developed, using feedback control, to counter the effects of uncertainty in the specific growth rate and thus determine an optimal operating strategy for pGH production. This policy was experimentally implemented in a 10 L fermenter and resulted in a 125% increase in productivity over the previous best experimental result with this system—in spite of significant plant-model mismatch.     

An oligosaccharyltransferase from Leishmania major increases the N‐glycan occupancy on recombinant glycoproteins produced in Nicotiana benthamiana

N‐glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central‐protein complex facilitating the N‐glycosylation of proteins in the lumen of the endoplasmic reticulum (ER). Not all potential N‐glycosylation sites are recognized in vivo and the site occupancy can vary in different expression systems, resulting in underglycosylation of recombinant glycoproteins. To overcome this limitation in plants, we expressed LmSTT3D, a single‐subunit oligosaccharyltransferase from the protozoan Leishmania major transiently in Nicotiana benthamiana, a well‐established production platform for recombinant proteins. A fluorescent protein‐tagged LmSTT3D variant was predominately found in the ER and co‐located with plant oligosaccharyltransferase subunits. Co‐expression of LmSTT3D with immunoglobulins and other recombinant human glycoproteins resulted in a substantially increased N‐glycosylation site occupancy on all N‐glycosylation sites except those that were already more than 90% occupied. Our results show that the heterologous expression of LmSTT3D is a versatile tool to increase N‐glycosylation efficiency in plants.     

Influence of Exogenous Triiodothyronine (T<Subscript>3</Subscript>) on Fecal Shedding of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157 in Cattle

Fecal prevalence of Escherichia coli O157 in ruminants is highest in the summer months and decreases to low or undetectable levels in the winter. We hypothesize that the seasonal variation of this pathogen is a result of physiological responses within the host animal to changing day length. The thyroid is an endocrine gland known to respond to changing day length. Two experiments were conducted to determine if a hyperthyroid status would initiate fecal shedding of E. coli O157 in cattle during the winter when shedding is virtually nonexistent (winter experiment) or influence cattle actively shedding E. coli O157 (summer experiment). Yearling cattle were group-penned under dry-lot conditions, adjusted to a high concentrate ration, and randomly assigned to treatment: control (1 mL corn oil injected s.c. daily) or triiodothyronine (T₃; 1.5 mg suspended in corn oil injected s.c daily). Cattle were individually processed daily for collection of fecal and blood samples. Treatment with exogenous T₃ produced a significant change in serum thyroid hormone concentrations indicative of a hyperthyroid status in both experiments. No differences (P>0.10) were observed in fecal shedding of E. coli O157 in the winter experiment. In the summer experiment, fecal shedding of E. coli O157 was decreased (P=0.05) by administration of T₃ during the treatment period (days 1–10), tended to be lower (P=0.08) during the following 7-day period of no treatment, and was lower (P=0.01) when examined across the entire experimental period. Results of this research indicate that the thyroid or its hormones may be involved in the seasonal shedding patterns of E. coli O157 in cattle.     

Cloning,expression and immunogenicity of ferric enterobactin binding protein Fep B from <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7

The gene coding for ferric enterobactin binding protein from E. coli O157:H7 was amplifi ed. This gene was cloned and expressed as C-terminal His (6)-tagged protein. The SDS-PAGE analysis of the total protein revealed only two distinct bands, with molecular masses of 31kDa and 34kDa. The Ni-NTA chromatography purifi ed FepB and the osmotically shocked periplasmic fraction of IPTG induced cells showed only a single band of 31 kDa. Polyclonal mouse antibody was raised against the recombinant protein during 4 weeks after immunization. Western blot analysis of the recombinant FepB with mouse antiserum revealeda single band of 31 kDa. Identification and purification of FepB helped reveal its appropriate molecular mass. Polyclonal antibody raised against the recombinant protein reacted with bacterial FepB. The recombinant protein FepB could have a protective effect against E. coli O157:H7 and might be useful as an effective vaccine.     

The history and evolution of Escherichia coli O157 and other Shiga toxin-producing E. coli

Shiga toxin-producing Escherichia coli (STEC) O157 is a formidable human pathogen with the capacity to cause large outbreaks of gastrointestinal illness. The known virulence factors of this organism are encoded on phage, plasmid and chromosomal genes. There are also likely to be novel, as yet unknown virulence factors in this organism. Many of these virulence factors have been acquired by E. coli O157 by transfer from other organisms, both E. coli and non-E. coli species. By examination of biochemical and genetic characteristics of various E. coli O157 strains and the relationships with other organisms, an evolutionary pathway for development of E. coli O157 as a pathogen has been proposed. E. coli O157 evolved from an enteropathogenic E. coli ancestor of serotype O55:H7, which contained the locus of enterocyte effacement containing the adhesin intimin. During the evolutionary process, Shiga toxins, the pO157 plasmid and other characteristics which enhanced virulence were acquired and other functions such as motility, sorbitol fermentation and β-glucuronidase activity were lost by some strains. It is likely that E. coli O157 is constantly evolving, and changes can be detected in genetic patterns during the course of infection. A variety of mechanisms may be responsible for the development of the virulent phenotype that we see today. Such changes include uptake of as yet uncharacterised virulence factors, possibly enhanced by a mutator phenotype, recombination within virulence genes to produce variant genes with different properties, loss of large segments of DNA (black holes) to enhance virulence and possible adaptation to different hosts. Although little is known about the evolution of non-O157 STEC it is likely that the most virulent clones evolved in a similar manner to E. coli O157. This revised version was published online in November 2006 with corrections to the Cover Date.     

Experimental infection in calves with a specific subtype of verocytotoxin-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 of bovine origin

Background  

In Sweden, a particular subtype of verocytotoxin-producing Escherichia coli (VTEC) O157:H7, originally defined as being of phage type 4, and carrying two vtx ₂ genes, has been found to cause the majority of reported human infections during the past 15 years, including both sporadic cases and outbreaks. One plausible explanation for this could be that this particular subtype is better adapted to colonise cattle, and thereby may be excreted in greater concentrations and for longer periods than other VTEC O157:H7 subtypes.     

Antimicrobial efficacy of lactoferrin, its amidated and pepsin-digested derivatives, and their combinations, on Escherichia coli O157:H7 and Serratia liquefaciens

Aims: To evaluate the in vitro bactericidal efficacy of lactoferrin (LF), its amidated (AMILF) and pepsin‐digested (PDLF) derivatives, and their combinations, on Escherichia coli O157:H7 and Serratia liquefaciens. Methods and Results: PDLF exhibited the most potent bactericidal efficacy on E. coli O157:H7 (>2·5 log₁₀ CFU ml^?1 reduction at concentrations ≥1 mg ml^?1), and AMILF on Ser. liquefaciens (1 log₁₀ CFU ml^?1 reduction at 0·25–0·50 mg ml^?1). Some combinations of LF with PDLF or AMILF showed a slight synergy on E. coli O157:H7 and Ser. liquefaciens. However, all combinations of AMILF with PDLF were less active than the sum of the individual effects of the two antimicrobials. Production of capsular polysaccharide by bacteria might be involved in antimicrobial resistance. Conclusions: Escherichia coli O157:H7 and Ser. liquefaciens showed marked differences in the sensitivity to LF and its derivatives. E. coli O157:H7 was strongly inhibited by PDLF, whereas the effect of LF and its derivatives on Ser. liquefaciens was weak to negligible. Significance and Impact of the Study: PDLF was the most promising of the tested antimicrobials on E. coli O157:H7. However, the resistance of Ser. liquefaciens to LF and its derivatives hinders their use in the food industry.