In vivo phosphorylation of a peptide tag for protein purification

Objectives

To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag.

Results

The level of the co-expressed CKIIα was controlled by the arabinose promoter and optimal phosphorylation was obtained with 2 % (w/v) arabinose as inductor. The effectiveness of the phosphorylation system was demonstrated by electrophoretic mobility shift assay (NUT-PAGE) and staining with a specific phosphoprotein-staining gel. The resulting phosphorylated tag was also used to purify the phosphoprotein by immobilized metal affinity chromatography, which relies on the specific interaction of phosphate moieties with Fe(III).

Conclusion

The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.

     

Production of single chain Fab (scFab) fragments in Bacillus megaterium

Background

The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli.

Results

The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli.

Conclusion

B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.     

Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions

Background

Eukaryotic ubiquitin and SUMO are frequently used as tags to enhance the fusion protein expression in microbial host. They increase the solubility and stability, and protect the peptides from proteolytic degradation due to their stable and highly conserved structures. Few of prokaryotic ubiquitin-like proteins was used as fusion tags except ThiS, which enhances the fusion expression, however, reduces the solubility and stability of the expressed peptides in E. coli. Hence, we investigated if MoaD, a conserved small sulfur carrier in prokaryotes with the similar structure of ubiquitin, could also be used as fusion tag in heterologous expression in E. coli.

Results

Fusion of MoaD to either end of EGFP enhanced the expression yield of EGFP with a similar efficacy of ThiS. However, the major parts of the fusion proteins were expressed in the aggregated form, which was associated with the retarded folding of EGFP, similar to ThiS fusions. Fusion of MoaD to insulin chain A or B did not boost their expression as efficiently as ThiS tag did, probably due to a less efficient aggregation of products. Interestingly, fusion of MoaD to the murine ribonuclease inhibitor enhanced protein expression by completely protecting the protein from intracellular degradation in contrast to ThiS fusion, which enhanced degradation of this unstable protein when expressed in E. coli.

Conclusions

Prokaryotic ubiquitin-like protein MoaD can act as a fusion tag to promote the fusion expression with varying mechanisms, which enriches the arsenal of fusion tags in the category of insoluble expression.     

Expression of adenylate kinase fused MEK1R4F in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application in ERK phosphorylation

Objective

To construct a highly expressed and active MEK1R4F (a constitutively active form of mitogen-activated protein kinase kinase 1) by fusion of soluble adenylate kinase (Adk) tag, resulting in Adk-MEK1R4F protein suitable for preparation of phosphorylated ERK.

Results

We fused the Adk to the N-terminus of MEK1R4F through overlapping PCR. The expression of Adk-MEK1R4F fusion protein increased ~10-fold in Escherichia coli, and was purified to 95% via two purification steps including Ni–NTA and Q Sepharose fast flow (QFF) chromatography. The purified Adk-MEK1R4F protein was functional for ERK phosphorylation and could use ADP in addition to ATP. The Adk-MEK1R4F had higher catalytic activity than regular MEK1R4F both in vitro and in cell-free extracts system.

Conclusions

Adenylate kinase was used as a soluble tag to facilitate MEK1R4F protein expression and its application in large-scale phosphorylated ERK1/2 preparation and purification.

     

Daily temperature profiles in and around Western Kenyan larval habitats of Anopheles gambiae as related to egg mortality

Background

Erythrocyte invasion by Plasmodium falciparum parasites represents a key mechanism during malaria pathogenesis. Erythrocyte binding antigen-181 (EBA-181) is an important invasion protein, which mediates a unique host cell entry pathway. A novel interaction between EBA-181 and human erythrocyte membrane protein 4.1 (4.1R) was recently demonstrated using phage display technology. In the current study, recombinant proteins were utilized to define and characterize the precise molecular interaction between the two proteins.

Methods

4.1R structural domains (30, 16, 10 and 22 kDa domain) and the 4.1R binding region in EBA-181 were synthesized in specific Escherichia coli strains as recombinant proteins and purified using magnetic bead technology. Recombinant proteins were subsequently used in blot-overlay and histidine pull-down assays to determine the binding domain in 4.1R.

Results

Blot overlay and histidine pull-down experiments revealed specific interaction between the 10 kDa domain of 4.1R and EBA-181. Binding was concentration dependent as well as saturable and was abolished by heat denaturation of 4.1R.

Conclusion

The interaction of EBA-181 with the highly conserved 10 kDa domain of 4.1R provides new insight into the molecular mechanisms utilized by P. falciparum during erythrocyte entry. The results highlight the potential multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasite's life cycle.     

Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection

Background

Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein.

Results

In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody.

Conclusion

The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material.     

Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species

Background

The ability to react early to possible outbreaks of Escherichia coli O157:H7 and to trace possible sources relies on the availability of highly discriminatory and reliable techniques. The development of methods that are fast and has the potential for complete automation is needed for this important pathogen.

Methods

In all 73 isolates of shiga-toxin producing E. coli O157 (STEC) were used in this study. The two available fully sequenced STEC genomes were scanned for tandem repeated stretches of DNA, which were evaluated as polymorphic markers for isolate identification.

Results

The 73 E. coli isolates displayed 47 distinct patterns and the MLVA assay was capable of high discrimination between the E. coli O157 strains. The assay was fast and all the steps can be automated.

Conclusion

The findings demonstrate a novel high discriminatory molecular typing method for the important pathogen E. coli O157 that is fast, robust and offers many advantages compared to current methods.     

A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines

Background

To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity.

Results

Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia.

Conclusions

The method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.     

Evolutionary conservation of domain-domain interactions

Background

Recently, there has been much interest in relating domain-domain interactions (DDIs) to protein-protein interactions (PPIs) and vice versa, in an attempt to understand the molecular basis of PPIs.

Results

Here we map structurally derived DDIs onto the cellular PPI networks of different organisms and demonstrate that there is a catalog of domain pairs that is used to mediate various interactions in the cell. We show that these DDIs occur frequently in protein complexes and that homotypic interactions (of a domain with itself) are abundant. A comparison of the repertoires of DDIs in the networks of Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens shows that many DDIs are evolutionarily conserved.

Conclusion

Our results indicate that different organisms use the same 'building blocks' for PPIs, suggesting that the functionality of many domain pairs in mediating protein interactions is maintained in evolution.     

The genome sequence of the protostome Daphnia pulex encodes respective orthologues of a neurotrophin, a Trk and a p75NTR: Evolution of neurotrophin signaling components and related proteins in the bilateria

Background

Neurotrophins and their Trk and p75NTR receptors play an important role in the nervous system. To date, neurotrophins, Trk and p75NTR have only been found concomitantly in deuterostomes. In protostomes, homologues to either neurotrophin, Trk or p75NTR are reported but their phylogenetic relationship to deuterostome neurotrophin signaling components is unclear. Drosophila has neurotrophin homologues called Spätzles (Spz), some of which were recently renamed neurotrophins, but direct proof that these are deuterostome neurotrophin orthologues is lacking. Trks belong to the receptor tyrosine kinase (RTK) family and among RTKs, Trks and RORs are closest related. Flies lack Trks but have ROR and ROR-related proteins called NRKs playing a neurotrophic role. Mollusks have so far the most similar proteins to Trks (Lymnaea Trk and Aplysia Trkl) but the exact phylogenetic relationship of mollusk Trks to each other and to vertebrate Trks is unknown. p75NTR belongs to the tumor necrosis factor receptor (TNFR) superfamily. The divergence of the TNFR families in vertebrates has been suggested to parallel the emergence of the adaptive immune system. Only one TNFR representative, the Drosophila Wengen, has been found in protostomes. To clarify the evolution of neurotrophin signaling components in bilateria, this work analyzes the genome of the crustacean Daphnia pulex as well as new genetic data from protostomes.

Results

The Daphnia genome encodes a neurotrophin, p75NTR and Trk orthologue together with Trkl, ROR, and NRK-RTKs. Drosophila Spz1, 2, 3, 5, 6 orthologues as well as two new groups of Spz proteins (Spz7 and 8) are also found in the Daphnia genome. Searching genbank and the genomes of Capitella, Helobdella and Lottia reveals neurotrophin signaling components in other protostomes.

Conclusion

It appears that a neurotrophin, Trk and p75NTR existed at the protostome/deuterostome split. In protostomes, a "neurotrophin superfamily" includes Spzs and neurotrophins which respectively form two paralogous families. Trks and Trkl proteins also form closely related paralogous families within the protostomian RTKs, whereby Trkls are absent in deuterostomes. The finding of p75NTR in several protostomes suggests that death domain TNFR superfamily proteins appeared early in evolution.     

Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

Background

The low pH environment of the human stomach is lethal for most microorganisms; but not Escherichia coli, which can tolerate extreme acid stress. Acid resistance in E. coli is hierarchically controlled by numerous regulators among which are small noncoding RNAs (sncRNA).

Results

In this study, we individually deleted seventy-nine sncRNA genes from the E. coli K12-MG1655 chromosome, and established a single-sncRNA gene knockout library. By systematically screening the sncRNA mutant library, we show that the sncRNA GcvB is a novel regulator of acid resistance in E. coli. We demonstrate that GcvB enhances the ability of E. coli to survive low pH by upregulating the levels of the alternate sigma factor RpoS.

Conclusion

GcvB positively regulates acid resistance by affecting RpoS expression. These data advance our understanding of the sncRNA regulatory network involved in modulating acid resistance in E. coli.     

High Yield Recombinant Expression, Characterization and Homology Modeling of Two Types of Cis-epoxysuccinic Acid Hydrolases

The cis-epoxysuccinate hydrolases (CESHs), members of epoxide hydrolase, catalyze cis-epoxysuccinic acid hydrolysis to form d(?)-tartaric acid or l(+)-tartaric acid which are important chemicals with broad scientific and industrial applications. Two types of CESHs (CESH[d] and CESH[l], producing d(?)- and l(+)-tartaric acids, respectively) have been reported with low yield and complicated purification procedure in previous studies. In this paper, the two CESHs were overexpressed in Escherichia coli using codon-optimized genes. High protein yields by one-step purifications were obtained for both recombinant enzymes. The optimal pH and temperature were measured for both recombinant CESHs, and the properties of recombinant enzymes were similar to native enzymes. Kinetics parameters measured by Lineweaver?CBurk plot indicates both enzymes exhibited similar affinity to cis-epoxysuccinic acid, but CESH[l] showed much higher catalytic efficiency than CESH[d], suggesting that the two CESHs have different catalytic mechanisms. The structures of both CESHs constructed by homology modeling indicated that CESH[l] and CESH[d] have different structural folds and potential active site residues. CESH[l] adopted a typical ??/??-hydrolase fold with a cap domain and a core domain, whereas CESH[d] possessed a unique TIM barrel fold composed of 8 ??-helices and 8 ??-strands, and 2 extra short ??-helices exist on the top and bottom of the barrel, respectively. A divalent metal ion, preferred to be zinc, was found in CESH[d], and the ion was proved to be crucial to the enzymatic activity. These results provide structural insight into the different catalytic mechanisms of the two CESHs.     

Effects of antibacterial mineral leachates on the cellular ultrastructure, morphology, and membrane integrity of Escherichia coli and methicillin-resistant Staphylococcus aureus

Background

We have previously identified two mineral mixtures, CB07 and BY07, and their respective aqueous leachates that exhibit in vitro antibacterial activity against a broad spectrum of pathogens. The present study assesses cellular ultrastructure and membrane integrity of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli after exposure to CB07 and BY07 aqueous leachates.

Methods

We used scanning and transmission electron microscopy to evaluate E. coli and MRSA ultrastructure and morphology following exposure to antibacterial leachates. Additionally, we employed Bac light LIVE/DEAD staining and flow cytometry to investigate the cellular membrane as a possible target for antibacterial activity.

Results

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging of E. coli and MRSA revealed intact cells following exposure to antibacterial mineral leachates. TEM images of MRSA showed disruption of the cytoplasmic contents, distorted cell shape, irregular membranes, and distorted septa of dividing cells. TEM images of E. coli exposed to leachates exhibited different patterns of cytoplasmic condensation with respect to the controls and no apparent change in cell envelope structure. Although bactericidal activity of the leachates occurs more rapidly in E. coli than in MRSA, LIVE/DEAD staining demonstrated that the membrane of E. coli remains intact, while the MRSA membrane is permeabilized following exposure to the leachates.

Conclusions

These data suggest that the leachate antibacterial mechanism of action differs for Gram-positive and Gram-negative organisms. Upon antibacterial mineral leachate exposure, structural integrity is retained, however, compromised membrane integrity accounts for bactericidal activity in Gram-positive, but not in Gram-negative cells.     

Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries

Background

Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), were overexpressed as double-tagged gene fusions. Both were tagged with glutathione S-transferase (GST) at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D), and the other with five contiguous arginine residues (5R).

Results

Both fusion proteins were overexpressed in Escherichia coli and retained enzymatic activity. The fusions were designed so their surfaces were charged under enzyme reaction conditions, which allowed isolation and immobilization in a single step, through a simple capture with either an anionic or a cationic exchanger (Sepharose Q or Sepharose SP) that electrostatically bound the 5D or 5R tag. The introduction of double tags only marginally altered the affinity of the enzymes for their substrates, and the double-tagged proteins were enzymatically active in both soluble and immobilized forms. Combined use of the fusion proteins led to the production of N-acetyl-D-neuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc).

Conclusion

Double-tagged gene fusions were overexpressed to yield two enzymes that perform sequential steps in sialic acid synthesis. The proteins were easily immobilized via ionic tags onto ionic exchange resins and could thus be purified by direct capture from crude protein extracts. The immobilized, double-tagged proteins were effective for one-pot enzymatic production of sialic acid.     

Molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli in Riyadh: emergence of CTX-M-15-producing E. coli ST131

Background

The prevalence of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) has increased recently. The aim of this study was to further characterise and to assess the occurrence of ESBL-EC in Riyadh, to use pulsed field gel electrophoresis (PFGE) typing to investigate the epidemiology of ESBL-EC and to determine the prevalence of ST131 in ESBL-EC.

Methods

A total of 152 E. coli isolates were collected at a tertiary hospital in Riyadh from September 2010 to June 2011. Genotypic and phenotypic methods were used to characterise ESBLs. PFGE was used to determine genetic relatedness. Detection of ST131 and CTX-M-like ESBLs was performed using real-time PCR.

Results

Of 152 strains, 31 were positive for ESBLs by phenotypic methods. The bla _CTX-M-15 gene was highly prevalent (30/31 strains, 96.77%) among the 31 ESBL-positive E. coli strains. The bla _CTX-M-27 gene was detected in one strain. Twenty (64.5%) out of 31 of ESBL-EC were ST131. PFGE revealed 29 different pulsotypes.

Conclusions

Our study documented the high prevalence of ESBLs in E. coli isolates, with CTX-M-15 as the predominant ESBL gene. ST131 clone producing CTX-M-15 has a major presence in our hospital. The high prevalence of CTX-M producers was not due to the spread of a single clone. To the best of our knowledge, this study represents the first report of CTX-M-15 and CTX-M-27 β-lactamases and the detection of the ST131 clone in Saudi E. coli isolates.     

Expression of Active Recombinant Human Tissue-Type Plasminogen Activator by Using In Vivo Polyhydroxybutyrate Granule Display

Recombinant human tissue plasminogen activator (rPA) is a truncated version of tissue plasminogen activator (tPA), which contains nine disulfide bonds and is prone to forming inactive inclusion bodies when expressed in bacteria. To obtain functional rPA expression, we displayed the rPA on the surface of polyhydroxybutyrate (PHB) granules using phasin as the affinity tag. rPA was fused to the N terminus of the phasin protein with a thrombin cleavage site as the linker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis showed that rPA fusion was successfully displayed on the surface of PHB granules. An activity assay indicated that the rPA fusion is active. The in vivo surface display strategy for functional rPA expression in Escherichia coli is distinct for its efficient folding and easier purification and may be expanded to the expression of other eukaryotic proteins with complex conformation.Tissue plasminogen activator (tPA) derives from a fibrinolytic system of blood vessel endothelial cells, activates plasminogen to form plasmin, and is an effective drug for thrombolytic therapy. Native tPA is composed of 527 amino acid residues with five structural domains and 17 disulfide bonds (19). Recombinant human tissue plasminogen activator (rPA) is a variant version of tPA with nine disulfide bonds, consisting of kringle 2 and serine protease domain (12). rPA was confirmed to possess enhanced capability for thrombolysis compared with that of tPA. Therefore, rPA is more beneficial for the treatment of acute myocardial infarction (17, 26, 28).Heterologous expression of tPA as well as rPA in Escherichia coli often results in the formation of the insoluble aggregates known as inclusion bodies due to the multidisulfide bonds (3). The refolding of the inclusion bodies in vitro is a long and difficult task, especially for proteins with complex conformation and multiple disulfide bonds. In order to obtain directly the functional rPA from recombinant E. coli, many approaches have been utilized: expressing the rPA gene in E. coli trxB gor ahpC* mutant strains, of which the cytoplasm is highly oxidized; fusing the rPA gene with gpIII of ΦM13 and linking to the OmpA signal sequence, through which rPA is secreted into the medium; exploiting the novel twin-arginine translocation (Tat) pathway to obtain active rPA in the periplasmic space based on its inherent properties; and cosecreting of rPA with chaperones and adding low-molecular-size medium additives to promote the formation of disulfide bonds (6, 11, 15, 25). However, the successful expression of rPA in its soluble or active form gives rise to another task: separation and purification of soluble active rPA from large amounts of other proteins in cytoplasm or medium.Normal protein purification typically involves several chromatographic steps. Each step can be costly and time-consuming (4). The development of simple and reliable methods for protein purification, which can be applied to arbitrary products, is therefore an important goal in bioseparation technology developments. One method that was recently developed is the addition of an affinity tag sequence to the target protein gene (13). It was demonstrated that heterologous proteins can be displayed actively on the surface of biopolyester granules in E. coli by fusing to the polyhydroxyalkanoate (PHA) synthase (PhaC), which serves as an affinity tag of PHA granules (21). PHA granules are carbon inclusions produced intracellularly by bacteria for coping with changing, often oligotrophic environments (1). These inclusions are composed of a hydrophobic polyester core and hydrophilic phospholipid membrane with many embedded proteins (24). Besides PhaC, phasins (namely PhaPs) are the main proteins tightly attached to the surface of polyhydroxybutyrate (PHB) granules, which can stabilize and prevent coalescence of separate PHB granules (22). Due to the inherent properties, PhaP has been used as the affinity tag in vivo to display recombinant proteins on the surface of PHB granules (5).In this study, we fused the rPA gene to the N terminus of phaP. A thrombin cleavage site was introduced between them to release rPA from PHB granules. The fusion gene was then expressed in engineered E. coli, which was conferred with the PHB production pathway by cloning the PHB biosynthesis genes. We confirmed that recombinant rPA fusion was able to be actively expressed in vivo on the surface of PHB granules.     

A study on the effect of different rifle calibres in euthanisation of grey seals (Halichoerus grypus) in seal traps in the Baltic Sea

Background

The already high and increasing occurrence of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli in European broiler populations is of concern due to the fact that third and fourth generation cephalosporins are deemed critically important in human medicine. In Sweden 34% of the broilers carry ESBL/pAmpC producing E. coli in their gut, despite the absence of a known selection pressure such as antimicrobial usages. The aim of the current study was to characterise a selection of E. coli strains carrying the bla _CTX-M-1, to determine if the spread was due to a specific clone.

Findings

Ten isolates carrying bla _CTX-M-1 from Swedish broilers belonged to eight different multi-locus sequence types with three isolates belonging to ST155. The ST155 isolates were identical as assessed by PFGE. The bla _CTX-M-1 was in all isolates carried on a plasmid of replicon type incI, which also transferred resistance to tetracycline and sulfamethoxazole.

Conclusion

The occurrence of ESBL-producing E. coli in the Swedish broilers is not due to the emergence of a single clone, but rather the spread of a specific incI plasmid carrying bla _CTX-M-1.