Prevalence of <Emphasis Type="Italic">Campylobacter</Emphasis> spp. in poultry and poultry products for sale on the Bulgarian retail market

The aim of this study was to investigate the presence of Campylobacter spp. in poultry and poultry products available for the consumers at retail markets in Bulgaria. Samples (n = 210) of poultry carcasses and poultry products for sale at the retail market in Bulgaria were analysed for the presence of Campylobacter spp., of these 35 frozen whole carcasses, 135 chilled poultry cuts (45 wing cuts, 45 thigh cuts and 45 fillet) and 40 thermally treated (ready-to-eat) poultry products. The results obtained showed that 35.2% of the frozen poultry carcasses for sale in the markets were Campylobacter contaminated. In the chilled poultry cuts Campylobacter was isolated at the highest percentage in wing- and thigh cuts, 91.1% and 88.9%, respectively. The fillet samples were contaminated by Campylobacter in 48.9% of cases. In the chilled poultry products as well as in the frozen carcasses C. jejuni (74.8%/70.3%) was the most commonly isolated Campylobacter species, with the remainder being C. coli (25.2%/29.7%). Campylobacter spp. were not detected in the thermally treated poultry products.     

Engineering alternative butanol production platforms in heterologous bacteria

Alternative microbial hosts have been engineered as biocatalysts for butanol biosynthesis. The butanol synthetic pathway of Clostridium acetobutylicum was first re-constructed in Escherichia coli to establish a baseline for comparison to other hosts. Whereas polycistronic expression of the pathway genes resulted in the production of 34 mg/L butanol, individual expression of pathway genes elevated titers to 200 mg/L. Improved titers were achieved by co-expression of Saccharomyces cerevisiae formate dehydrogenase while overexpression of E. coli glyceraldehyde 3-phosphate dehydrogenase to elevate glycolytic flux improved titers to 580 mg/L. Pseudomonas putida and Bacillus subtilis were also explored as alternative production hosts. Polycistronic expression of butanol biosynthetic genes yielded butanol titers of 120 and 24 mg/L from P. putida and B. subtilis, respectively. Production in the obligate aerobe P. putida was dependent upon expression of bcd-etfAB. These results demonstrate the potential of engineering butanol biosynthesis in a variety of heterologous microorganisms, including those cultivated aerobically.     

Penetration of poultry meat byPseudomonas andLactobacillus spp.

The penetration of poultry muscle strips byPseudomonas putida, P. fragi, Lactobacillus plantarum andL. casei was studied at 37°C. The first three bacteria penetrated along the muscular septa whileLactobacillus casei did not invade the poultry muscle strips at all. The penetration by both pseudomonads occurred throughout the 3 cm muscle strip butL. plantarum showed minor penetration up to 2 cm only.     

Heterologous expression of the<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferase genes in<Emphasis Type="Italic"> Clostridium acetobutylicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis> for the production of isoamyl acetate

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production.     

Modeling of the pyruvate production with<Emphasis Type="Italic"> Escherichia coli</Emphasis> in a fed-batch bioreactor

A family of 10 competing, unstructured models has been developed to model cell growth, substrate consumption, and product formation of the pyruvate producing strain Escherichia coli YYC202 ldhA::Kan strain used in fed-batch processes. The strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate (using glucose as the carbon source) resulting in an acetate auxotrophy during growth in glucose minimal medium. Parameter estimation was carried out using data from fed-batch fermentation performed at constant glucose feed rates of q_VG=10 mL h^–1. Acetate was fed according to the previously developed feeding strategy. While the model identification was realized by least-square fit, the model discrimination was based on the model selection criterion (MSC). The validation of model parameters was performed applying data from two different fed-batch experiments with glucose feed rate q_VG=20 and 30 mL h^–1, respectively. Consequently, the most suitable model was identified that reflected the pyruvate and biomass curves adequately by considering a pyruvate inhibited growth (Jerusalimsky approach) and pyruvate inhibited product formation (described by modified Luedeking–Piret/Levenspiel term).List of symbols c_A acetate concentration (g L^–1) - c_A,0 acetate concentration in the feed (g L^–1) - c_G glucose concentration (g L^–1) - c_G,0 glucose concentration in the feed (g L^–1) - c_P pyruvate concentration (g L^–1) - c_P,max critical pyruvate concentration above which reaction cannot proceed (g L^–1) - c_X biomass concentration (g L^–1) - K_I inhibition constant for pyruvate production (g L^–1) - K_I^A inhibition constant for biomass growth on acetate (g L^–1) - K_P saturation constant for pyruvate production (g L^–1) - K_P inhibition constant of Jerusalimsky (g L^–1) - K_S^A Monod growth constant for acetate (g L^–1) - K_S^G Monod growth constant for glucose (g L^–1) - m_A maintenance coefficient for growth on acetate (g g^–1 h^–1) - m_G maintenance coefficient for growth on glucose (g g^–1 h^–1) - n constant of extended Monod kinetics (Levenspiel) (–) - q_V volumetric flow rate (L h^–1) - q_VA volumetric flow rate of acetate (L h^–1) - q_VG volumetric flow rate of glucose (L h^–1) - r_A specific rate of acetate consumption (g g^–1 h^–1) - r_G specific rate of glucose consumption (g g^–1 h^–1) - r_P specific rate of pyruvate production (g g^–1 h^–1) - r_P,max maximum specific rate of pyruvate production (g g^–1 h^–1) - t time (h) - V reaction (broth) volume (L) - Y_P/G yield coefficient pyruvate from glucose (g g^–1) - Y_X/A yield coefficient biomass from acetate (g g^–1) - Y_X/A,max maximum yield coefficient biomass from acetate (g g^–1) - Y_X/G yield coefficient biomass from glucose (g g^–1) - Y_X/G,max maximum yield coefficient biomass from glucose (g g^–1) - growth associated product formation coefficient (g g^–1) - non-growth associated product formation coefficient (g g^–1 h^–1) - specific growth rate (h^–1) - _max maximum specific growth rate (h^–1)     

Enhanced production of recombinant protein inEscherichia coli using silkworm hemolymph

The effect of silkworm hemolymph on the expression of recombinant protein inEscherichia coli was investigated. The addition of silkworm hemolymph to the culture medium increased the production of recombinant β-galactosidase inE. coli. The production was dependent on the concentration of the added silkworm hemolymph, which increased 2-, 5-, and 8-fold in media supplemented with 1,3, and 5% silkworm hemolymph, respectively. To identify the effective component, the silkworm hemolymph was fractionated by gel filtration column chromatography. A fraction, with a molecular weight of about 30 K was identified as the effective component.     

Norepinephrine-dependently released Dr fimbriae of diffusely adhering Escherichia coli strain IH11128 promotes a mitogen-activated protein kinase ERK1/2-dependent production of pro-inflammatory cytokine, IL-8 in human intestinal Caco-2/TC7 cells

The diffusely adhering Escherichia coli (Afa/Dr DAEC) are associated with recurrent urinary tract infections in adults as well as with diarrheal disease in infants. We previously demonstrated that in wild-type strain IH11128, the Dr fimbriae is released in the extracellular medium in response to multiple environmental signals such as temperature, low aeration and rich medium. A number of molecules of eukaryotic origin, such as catecholamines, have been reported to stimulate bacterial growth and virulence factor production. We show that norepinephrine affects the production and release of Dr fimbriae in Afa/Dr DAEC WT-IH11128 bacteria. The regulatory mechanism involved with norepinephrine-induced Dr fimbriae liberation was apparently due to a differential induction of genes draC, encoding the usher, and draE, encoding the major fimbrial subunit. In addition, we show that the released Dr fimbriae induces the phosphorylation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 (ERK1/2) and the production of the pro-inflammatory cytokine, IL-8 in fully differentiated cultured human intestinal Caco-2/TC7 cells.     

Pilot-scale production and purification of a staphylokinase-based fusion protein over-expressed in Escherichia coli

SFH, a recombinant staphylokinase-based fusion protein linked by the factor Xa recognition peptide at the N-terminus of hirudin, is a promising therapeutic candidate for thromboembolic diseases. To develop SFH into a new thrombolytic agent, scaled-up production was carried out to provide sufficient preparation for animal safety and clinical studies. Here, we describe a pilot-scale cultivation and purification process for the production of SFH. A high-cell-density fed-batch cultivation for the production of SFH in E. coli was developed in a 40-L bioreactor, which produced about 1.1 g/L of recombinant protein. SFH was purified to homogeneity from the E. coli lysate by expanded bed adsorption chromatography and anion-exchange chromatography, with over 99% purity and 54% recovery. Moreover, the residual endotoxin content was less than 0.5 EU/mL. The molecular weight and in vitro bioactivity of SFH were also determined by electrospray ionization-mass spectrometry (ESI-MS) and fibrinolytic activity assay, respectively.     

Evaluation of Beauveria bassiana applications against adult house fly, Musca domestica, in commercial caged-layer poultry facilities in New York state

Applications of a commercially produced Beauveria bassiana product, balEnce, were compared with pyrethrin treatments for the control of adult house flies in New York high-rise, caged-layer poultry facilities. An integrated fly management program, which included the release of house fly pupal hymenopteran parasitoids, was used at all facilities. Adult house fly populations were lower in B. bassiana-treated facilities during the spray and post-spray periods, as recorded on spot cards. Concurrently, the numbers of house fly larvae recovered in B. bassiana-treated facilities were less than one-half that of the pyrethrin-treated facilities. House fly pupal parasitism levels were low, but similar under both treatment regimes. The numbers of adult and larval Carcinops pumilio, a predatory beetle, recovered from B. bassiana-treated facilities were 43 and 66% greater than from the pyrethrin-treated facilities, respectively.     

Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli

The increased synthesis of building blocks of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) through metabolic engineering is a way to enhance the production of carotenoids. Using E. coli as a host, IPP and DMAPP supply can be increased significantly through the introduction of foreign MVA (mevalonate) pathway into it. The MVA pathway is split into two parts with the top and bottom portions supplying mevalonate from acetyl-CoA, and IPP and DMAPP from mevalonate, respectively. The bottom portions of MVA pathway from Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Saccharomyces cerevisiae were compared with exogenous mevalonate supplementation for β-carotene production in recombinant Escherichia coli harboring β-carotene synthesis genes. The E. coli harboring the bottom MVA pathway of S. pneumoniae produced the highest amount of β-carotene. The top portions of MVA pathway were also compared and the top MVA pathway of E. faecalis was found out to be the most efficient for mevalonate production in E. coli. The whole MVA pathway was constructed by combining the bottom and top portions of MVA pathway of S. pneumoniae and E. faecalis, respectively. The recombinant E. coli harboring the whole MVA pathway and β-carotene synthesis genes produced high amount of β-carotene even without exogenous mevalonate supplementation. When comparing various E. coli strains – MG1655, DH5α, S17-1, XL1-Blue and BL21 – the DH5α was found to be the best β-carotene producer. Using glycerol as the carbon source for β-carotene production was found to be superior to glucose, galactose, xylose and maltose. The recombinant E. coli DH5α harboring the whole MVA pathway and β-carotene synthesis genes produced β-carotene of 465 mg/L at glycerol concentration of 2% (w/v).     

Bacterial-based membrane protein production

Escherichia coli is by far the most widely used bacterial host for the production of membrane proteins. Usually, different strains, culture conditions and production regimes are screened for to design the optimal production process. However, these E. coli-based screening approaches often do not result in satisfactory membrane protein production yields. Recently, it has been shown that (i) E. coli strains with strongly improved membrane protein production characteristics can be engineered or selected for, (ii) many membrane proteins can be efficiently produced in E. coli-based cell-free systems, (iii) bacteria other than E. coli can be used for the efficient production of membrane proteins, and, (iv) membrane protein variants that retain functionality but are produced at higher yields than the wild-type protein can be engineered or selected for. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

An evolutionary strategy for isobutanol production strain development in Escherichia coli

Higher alcohols such as isobutanol possess several physical characteristics that make them attractive as biofuels such as higher energy densities and infrastructure compatibility. Here we have developed a rapid evolutionary strategy for isolating strains of Escherichia coli that effectively produce isobutanol from glucose utilizing random mutagenesis and a growth selection scheme. By selecting for mutants with the ability to grow in the presence of the valine analog norvaline, we obtained E. coli NV3; a strain with improved 24-h isobutanol production (8.0 g/L) in comparison with a productivity of 5.3 g/L isobutanol obtained with the parental wild type strain. Genomic sequencing of NV3 identified the insertion of a stop codon in the C-terminus of the RNA polymerase σ^s-factor, RpoS. Upon repair of this inhibitory mutation (strain NV3r1), a final isobutanol titer of 21.2 g/L isobutanol was achieved in 99 h with a yield of 0.31 g isobutanol/g glucose or 76% of theoretical maximum. Furthermore, a mutation in ldhA, encoding d-lactate dehydrogenase, was identified in NV3; however, repair of LdhA in NV3r1 had no affect on LdhA activity detected from cell extracts or on isobutanol productivity. Further study of NV3r1 may identify novel genotypes that confer improved isobutanol production.     

Sustainable coccidiosis control in poultry production: the role of live vaccines

The development of new methods of administering coccidiosis vaccines has facilitated their use in the hatchery and thereby improved prospects for the economic vaccination of broilers. The acquisition of protective immunity to Eimeria species is boosted by further exposure to infection after vaccination. Factors that affect the reproductive efficiency of non-attenuated and attenuated vaccines are considered and the key role that oocyst production plays in establishing and maintaining uniform immunity in a flock of chickens is discussed. In addition to immunisation, a possible advantage to the application of certain vaccines is that their use could repopulate poultry houses with drug-sensitive organisms. Theoretical rotation programmes in which the use of drugs is alternated with that of vaccines are described. Variability of the cross-protective immune response between strains of the same species should be considered during vaccine development and subsequent use. The significance of less common species of Eimeria, not included in all vaccines, also needs to be assessed. An important consideration is the occurrence of pathogens other than Eimeria (such as the bacterium Clostridium) in flocks given coccidiosis vaccines and the methods by which they might be controlled. More research is required into the relationship between bacterial and viral infections of poultry and coccidiosis vaccination. Vaccines need to be developed that are simple to apply and cost effective for use in areas of the world where small-scale poultry production is commonplace. In the near future it is likely that more live vaccines based upon oocysts derived from attenuated strains of Eimeria will be developed but in the longer term vaccines will be based on the selective presentation to the host of specific molecules that can induce protective immunity. This achievement will require significant investment from the private and public sectors, and, if successful, will facilitate the sustainable control of coccidiosis in poultry production.     

Molecular Identification of Aeromonas and Coliform Bacteria Isolated on m Endo Media from Lake Erie Waters

Summary Bacteria from recreational waters collected from two Lake Erie beaches in Dunkirk, New York were plated onto m Endo LES media. The 16S rRNA gene was then amplified from coliform and non-coliform bacteria using the polymerase chain reaction. The PCR products were characterized by restriction fragment length polymorphism (RFLP) analysis. A total of 8 RFLP groups were identified from the analysis of 920 samples and selected PCR products from each group were sequenced. The DNA sequence analysis indicated that more than half of the bacteria identified as coliforms on the m Endo plates belonged to the genus Aeromonas from the family Aeromonadaceae. Most of the remaining coliforms were from the Enterobacteriaceae. The data indicate that m Endo agar plates allow the growth of non-coliform bacteria, especially Aeromonas species.     

Over-expression in E. coli and purification of the human OCTN1 transport protein

The hOCTN1 amplified from skin fibroblast RNA was cloned in pET-28a(+) or in pH6EX3 plasmid. The encoded recombinant hOCTN1 resulted in a 6-His tagged fusion protein with a 34 or 21 amino acid extra N-terminal sequence in the pET-28a(+)-hOCTN1 or in the pH6EX3-hOCTN1 constructs, respectively. Both constructs were used to express the hOCTN1 in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained with the pH6EX3-hOCTN1 after 6 h of induction with IPTG at 28 °C. The expressed protein with an apparent molecular mass of 54 kDa, was collected in the insoluble fraction of the cell lysate. Further improvement was obtained using the E. coli RosettaGami2(DE3)pLysS strain to express the protein encoded by pH6EX3-hOCTN1. After 6 h of induction with IPTG at 28 °C, hOCTN1 accounted for 30% of the total protein in the insoluble pellet. This protein fraction was washed with Triton X-100 and deoxycholate, solubilized with a buffer containing 0.8% Sarkosyl, 3 M urea and applied to a Ni²⁺-chelating chromatography column. The homogeneously purified hOCTN1 was eluted with a buffer containing 50 mM imidazole, 0.1% Triton X-100 and 50 mM 2-mercaptoethanol. A yield of about 3 mg purified protein per liter of cell culture was obtained.     

Detection of Campylobacter jejuni in poultry samples using an enzyme-linked immunoassay coupled with an enzyme electrode

An enzyme-linked immunoassay coupled with a tyrosinase modified enzyme electrode was used for rapid detection of Campylobacter jejuni. The immunomagnetic separation (IMS) method was investigated to achieve optimal isolation of C. jejuni cells. Eight types of beads with three different sizes and function groups were coated with anti-C. jejuni to isolate C. jejuni from the sample solution. Bead size and coating methods were found to be major factors that influenced the capture efficacy. Streptavidin-labeled beads (2.8 μm) provided the greatest capture ability. Three blocking reagents were tested to minimize non-specific binding. Bovine serum albumin (BSA) showed the best blocking capability. Two IMS formats were tested. Competitive immunoassay cut the detection time to 1.5 h, but the detection limit was relatively high (10⁶ CFU/ml). This system was evaluated using C. jejuni pure culture and poultry samples inoculated with C. jejuni. This detection method for C. jejuni could be completed within 2.5 h and had a detection limit of 2.1×10⁴ CFU/ml. No significant difference was found between pure culture samples and poultry samples (P>0.01). A linear relationship was found between C. jejuni cell numbers and the peak current ratio in a range of 10²–10⁷ CFU/ml (R²=0.94).     

Comparison of poultry exudate and carcass rinse sampling methods for the recovery of Campylobacter spp. subtypes demonstrates unique subtypes recovered from exudate

The carcass rinse procedure is a method commonly used for the detection of Campylobacter spp. on processed poultry products. Alternatively, carcass exudate (weep or drip), a viscous fluid comprised of blood and water that leaks into packaging, can also be sampled. It is unknown however if direct carcass rinse or exudate/weep can be utilized to preferentially recover different Campylobacter spp. subtypes. If there is a difference in subtypes recovered, the Campylobacter spp. subtypes from carcass rinse analysis may not be indicative of consumer exposure, as the exudate is the fluid to which consumers are potentially exposed to due to kitchen cross-contamination. Experiments were conducted to determine if there are differences in recovery of Campylobacter spp. subtypes between the two methodologies. The experiment was performed in triplicate using three flocks located on different farms. For each flock, 50 fecal samples were obtained on the farm, 25 carcass rinses during pre-chill processing, 25 carcass rinses during post-chill processing, and 50 samples from exudate from carcasses stored at 4 degrees C (25 after 2-day storage and 25 after 6-day storage). Each sample type was cultured for Campylobacter spp. Isolates recovered from positive samples were subtyped using flaA SVR (flagellin A-short variable region) DNA sequence typing and compared for relatedness. The data demonstrated that multiple subtypes of Campylobacter jejuni were present in a flock, and that subtypes present in a flock during production were also present on the final processed product. Subtypes recovered by the two recovery methodologies were similar based on flaA SVR classification. Combining the totals from all 3 flocks a total of 10 flaA SVR subtypes were recovered from post-chill carcass rinses and 9 subtypes recovered from 6-day exudate samples.     

Defined competitive exclusion cultures in the prevention of enteropathogen colonisation in poultry and swine

A competitive exclusion culture (CE) containing a mixture of 29 different bacterial isolates obtained from the cecae of broiler chickens was developed utilizing continuous-flow culture techniques. This culture (CF3) has been efficacious in controlling gut colonization by enteropathogens in both experimentally infected broilers and under commercial field conditions. In day-old broiler chicks provided CF3, and challenged with 10,000 CFU Salmonella typhimurium greater than a 99% reduction in Salmonella cecal colonization levels was observed compared to control chicks. Similarly, CF3 has also been shown to protect experimentally infected broiler chicks from cecal colonization by S. enteritidis (Phage types 4 and 13), S. gallinarum, Listeria Monocytogenes, and Escherichia coli O157:H7. A commercial product was developed from CF3 and is sold under the tradename PREEMPT™. In a Food and Drug Administration approved, double blinded, pivotal field trial, chicks treated with PREEMPTT™ had significantly fewer salmonellae than untreated chicks at end-of-growout. This product is the first of its kind available to the U.S. poultry industry. Using similar technology a product has also been developed that decreases shedding of salmonellae in neonate and weaned pigs, and also has been shown to reduce mortality associated with enteropathogens in young pigs both in the laboratory and in a commercial swine herd. This revised version was published online in August 2006 with corrections to the Cover Date.     

Re-engineering Escherichia coli for ethanol production

A lactate producing derivative of Escherichia coli KO11, strain SZ110, was re-engineered for ethanol production by deleting genes encoding all fermentative routes for NADH and randomly inserting a promoterless mini-Tn5 cassette (transpososome) containing the complete Zymomonas mobilis ethanol pathway (pdc, adhA, and adhB) into the chromosome. By selecting for fermentative growth in mineral salts medium containing xylose, a highly productive strain was isolated in which the ethanol cassette had been integrated behind the rrlE promoter, designated strain LY160 (KO11, Δfrd::celY _Ec ΔadhE ΔldhA, ΔackA lacA::casAB _Ko rrlE::(pdc _Zm -adhA _Zm -adhB _Zm -FRT-rrlE) pflB ⁺ ). This strain fermented 9% (w/v) xylose to 4% (w/v) ethanol in 48 h in mineral salts medium, nearly equal to the performance of KO11 with Luria broth.