大肠杆菌中吡咯喹啉醌合成途径的构建

目前吡咯喹啉醌（Pyrroloquinoline quinone,PQQ）在大肠杆菌中的异源合成主要以质粒为表达载体进行,但是质粒载体难以进行合成途径多基因表达的系统优化,并且容易造成发酵不稳定。作者以大肠杆菌为底盘生物,利用CRISPR/Cas9基因编辑技术,在基因组水平系统优化PQQ的合成。将来源于Gluconobacter oxydans 621H的操纵子pqqABCDE引入底盘大肠杆菌,并进一步通过优化合成途径基因表达强度,敲除大肠杆菌自身抑制基因及强化PQQ的胞内需求与胞外转运等,获得了一株能够高效合成PQQ的工程菌,摇瓶发酵72 h时产量达到86.3 mg/L。以大肠杆菌为底盘构建PQQ高效合成途径的工作能够为后续以其他底盘生物生产PQQ及相关代谢产物提供借鉴。     

Inactivation of Escherichia coli in orange juice using ozone

This research investigated the efficacy of gaseous ozone for the inactivation of Escherichia coli ATCC 25922 and NCTC 12900 strains in orange juice. Orange juice inoculated with E. coli (10⁶ CFU mL^− 1) as a challenge microorganism was treated with ozone at 75–78 µg mL^− 1 for different time periods (0–18 min). The efficacy of ozone for inactivation of both strains of E. coli was evaluated as a function of different juice types: model orange juice, fresh unfiltered juice, juice without pulp, and juice filtered through 500 µm or 1 mm sieves. Fast inactivation rates for total reduction of E. coli were achieved in model orange juice (60 s) and in juice with low pulp content (6 min). However, in unfiltered juice inactivation was achieved after 15–18 min. This indicated that juice organic matter interferes with antibacterial activity of gaseous ozone. The effect of prior acid (pH 5.0) exposure of E. coli strains on the inactivation efficacy of ozone treatment was also investigated. There was a strain effect observed, where prior acid exposure resulted in higher inactivation times in some cases by comparison with the control cells. However, the overarching influence on inactivation efficacy of ozone was related to the pulp content. Generally, the applied gaseous ozone treatment of orange juice resulted in a population reduction of 5 log cycles.

Industrial relevance

To facilitate the preservation of unstable nutrients many juice processors have investigated alternatives to thermal pasteurisation, including un-pasteurised short shelf life juices with high retail value. This trend has continued within the European Union. However within the US recent regulations by the FDA have required processors to achieve a 5-log reduction in the numbers of the most resistant pathogens in their finished products. Pathogenic E. coli may survive in acid environments such as fruit juices for long periods. This study demonstrates that the use of ozone as a non-thermal technology is effective for inactivation of E. coli and acid exposed E. coli in orange juice. Information on the design of the ozone treatment for inactivation of E. coli which results into safe juice products is also among the main outputs of this work. Ozone auto-decomposition makes this technology safe for fruit juice processing.     

Inactivation effect of an 18-T pulsed magnetic field combined with other technologies on Escherichia coli

The inactivation effect of 18 T pulsed magnetic fields in combination with selected non-thermal technologies was studied on Escherichia coli ATCC 11775. The bacteria were subjected to a treatment of either ultrasound (20 kHz, 70 W, 242 μm), high hydrostatic pressure (207 MPa, 5 min), pulsed electric field (6.25 kV/cm, 5.6 ms), or anti-microbials (Nisin 77.5 mg/l, lysozyme 1 mg/ml) and 50 magnetic field pulses (18 T, 30 μs). No additional inactivation or cell damage due to exposure to the pulsed magnetic field at 42°C was observed.     

Effect of growth rate reduction and genetic modifications on acetate accumulation and biomass yields in Escherichia coli

Although acetate biosynthesis in Escherichia coli provides an important intermediary for ATP synthesis, its accumulation inhibits both cell growth and protein production. Since pyruvate provides the largest flux to acetate and is central to the problem of acetate production, acetate accumulation could be reduced or abolished if the pyruvate pool for the TCA cycle was reduced. To examine this possibility, various pyruvate kinase (pyk) and phosphotransferase system (pts) mutants were tested for acetate production in batch cultures with glucose as the only carbon source. The pykA pykF mutant exhibited significant reductions in the specific growth rate and acetate production compared with the wild-type strain. Interestingly, in the case of pts and pts pyk mutants in which increased biomass yields were observed in comparison with the wild-type strain, no acetate production was detected. Therefore, these mutants are potentially useful for higher production of recombinant proteins. The results from the continuous cultivation performed using the wild-type strain at various dilution rates, suggest acetate reduction as a consequence of both genetic changes and growth rate diminutions.     

Prevalence of tetracycline resistance and genotypic analysis of populations of Escherichia coli from animals, carcasses and cuts processed at a pig slaughterhouse

A Danish pig slaughterhouse was visited in this study to investigate the impact of carcass processing on prevalence of tetracycline-resistant Escherichia coli, and to identify the origins of carcass contaminations with E. coli by assessing genetic diversity of E. coli populations on carcasses. A total of 105 carcasses were sampled at five sequential stages: after stunning, after scalding, after splitting, after cooling and after cutting. Total and tetracycline-resistant E. coli were counted for each sample and tetracycline resistance prevalence per sample was calculated by the fraction of tetracycline-resistant E. coli out of total E. coli. From 15 repeatedly sampled carcasses, 422 E. coli isolates from faeces, stunned carcasses, split carcasses and chilled carcasses were examined by pulse-field gel electrophoresis (PFGE) and tested for antimicrobial susceptibility. The results showed that E. coli counts and the prevalence of tetracycline-resistant E. coli per sample were both progressively reduced after each sampling stage. PFGE analysis showed that E. coli populations from stunned carcasses were highly genetically diverse, compared with those from split carcasses and faeces. Thirteen carcasses (87%) were contaminated with E. coli that were also isolated from faeces of either the same or other pigs slaughtered on the same day; and 80% of stunned carcasses shared the same E. coli PFGE subtypes. The results suggest that some carcass processing steps in the slaughterhouse were effective in reducing both E. coli numbers and the tetracycline resistance prevalence in E. coli on carcasses. Faeces from the same or other pigs slaughtered on the same day were likely to be an important source of E. coli carcass contamination. Combined data from E. coli enumeration, PFGE typing and antimicrobial susceptibility testing suggest that tetracycline-susceptible E. coli probably persisted better compared to resistant ones during the carcass processing.     

Biofilm formation by Escherichia coli in hypertonic sucrose media

High osmotic environments produced by NaCl or sucrose have been used as reliable and traditional methods of food preservation. We tested, Escherichia coli as an indicator of food-contaminating bacterium, to determine if it can form biofilm in a hyperosmotic environment. E. coli K-12 IAM1264 did not form biofilm in LB broth that contained 1 M NaCl. However, the bacterium formed biofilm in LB broth that contained 1 M sucrose, although the planktonic growth was greatly suppressed. The biofilm, formed on solid surfaces, such as titer-plate well walls and glass slides, solely around the air–liquid interface. Both biofilm forming cells and planktonic cells in the hypertonic medium adopted a characteristic, fat and filamentous morphology with no FtsZ rings, which are a prerequisite for septum formation. Biofilm forming cells were found to be alive based on propidium iodide staining. The presence of 1 M sucrose in the food environment is not sufficient to prevent biofilm formation by E. coli.     

A microwire sensor for rapid detection of Escherichia coli K-12 in fresh produce

A rapid immunofluorescence method for foodborne pathogens in food systems using microwire sensor coupled with high frequency alternating current was developed. The method was intended to enrich and quantify E. coli cells internalized in baby spinach leaves. The targeted bacterial cells in the sample solution were captured on microwires in a diameter of 25 μm, and were bound to fluorescein isothiocyanate (FITC) labeled polyclonal E. coli antibodies. Fluorescent images of the FITC antibodies were obtained using a fluorescence microscope equipped with a charge-coupled-device (CCD) camera, and the fluorescent intensity (FI) was quantified through image processing. The capture of E. coli K-12 in PBS buffer was optimized when the electric field was generated at the frequency of 3 MHz and 20 Vpp with bacterial concentration of 10⁷ CFU/mL. The detection limit of our sensing device was determined to be 10³ CFU/mL. Field emission scanning electron microscopy (FESEM) was used to validate and visualize E. coli cells captured on the tip surface. The sensitivity and specificity of the developed sensor has been successfully validated by testing E. coli internalized in baby spinach leaves. The immunofluorescence detection has been completed within 15 min. Moreover, it was found that the enrichment process of E. coli cells using the dielectrophoretic force was rarely affected by food particles, which proved the sensing selectivity. The developed sensor is expected to meet the steady demand for a simple, rapid, highly sensitive detection approach to quantify the targeted microbes in food systems.

Industrial Relevance

There has been an increase in the number of foodborne illnesses linked to the consumption of fresh and minimally processed fruits and vegetables. Some E.coli strains such as E.coli O 157:H7, can cause a variety of diseases, including diarrhea, urinary tract infections, respiratory diseases, meningitis and more. In general, consumers wash the fresh produces under cold running tap water to remove any lingering dirt on the surface of the produces before eating or preparing. However, how do the consumers know if there is any possible pathogen hiding inside of the fresh produce after rinsing? It was reported from many researchers that, the E.coli internalization, which may occur when fresh produces intake E. coli containing water or manure from the soil, would be a main cause of the foodborne illness outbreak. To ensure the safety of drinking water, E.coli concentration cannot be higher than 1 CFU/mL. How can we detect such a low level of E.coli in an easy yet efficient way? To our knowledge, none of the traditional detecting approaches such as cultural based method, polymerase chain reaction(PCR), surface plasmon resonance (SPR) biosensor, and Latex Agglutination, has performed perfectly. Hence, a rapid and accurate technique for detecting foodborne pathogens in fresh produce is urgently needed in order to secure the food safety.To overcome this issue, a simple detection method for foodborne pathogens in food systems using the microwire sensor coupled with high frequency alternative current was developed. The sensitivity and specificity of the developed sensor have been successfully validated by testing with E. coli internalized inside baby spinach leaves. It was found that spinach particles rarely affect the performance of our sensing device, which shows a promising prospect of its application in food industries.     

UV-C inactivation of Escherichia coli at different temperatures

The influence of treatment parameters (dose and temperature), treatment medium characteristics (absorption coefficient, pH and water activity) and microbiological factors (strain, growth phase and UV damage and repair capacity) on Escherichia coli UV-C resistance has been investigated. UV-C doses to inactivate at 25 °C 99.99% of the initial population (4D) of five strains of E. coli in McIlvaine buffer of pH 7.0 with tartrazine added (absorption coefficient of 10.77 cm⁻¹) were 16.60, 14.36, 14.36, 13.22, 11.18 J/mL for strains E. coli STCC 4201, STCC 471, STCC 27325, O157:H7 and ATCC 25922, respectively. The entrance in the stationary growth phase increased the 4D value of the most resistant strain, E. coli STCC 4201, from 13.09 to 17.23 J/mL. Survivors to UV treatments showed neither oxidative damages nor injuries in cell envelopes. On the contrary, the photoreactivation by the incubation of plates for 60 min below visible light (11.15 klx) increased the dose to 18.97 J/mL. The pH and the water activity of the treatment medium did not affect the UV tolerance of E. coli STCC 4201, but the lethal effect of the treatments decreased exponentially (Log₁₀4D = − 0.0628α + 0.624) by increasing the absorption coefficient (α). A treatment of 16.94 J/mL reached 6.35, 4.35, 2.64, 1.93, 1.63, 1.20, 1.02 and 0.74 Log₁₀ cycles of inactivation with absorption coefficients of 8.56, 10.77, 12.88, 14.80, 17.12, 18.51, 20.81 and 22.28 cm⁻¹. The temperature barely changed the UV resistance up to 50.0 °C. Above this threshold, inactivation rates due to the combined process synergistically increased with the temperature. The magnitude of the synergism decreased over 57.5 °C. An UV treatment of 16.94 J/mL in media with an absorption coefficient of 22.28 cm⁻¹ reached 1.23, 1.64, 2.36, 4.01 and 6.22 Log₁₀ cycles of inactivation of E. coli STCC 4201 at 50.0, 52.5, 55.5, 57.5 and 60.0 °C, respectively.

Industrial relevance

Results obtained in this investigation show that UV light applied at mild temperatures (57.5 to 60 °C) could be an alternative to heat treatments for 5-Log₁₀ reductions of E. coli in liquid foods. Since microbial resistance to UV-C light did not depend on the pH and water activity (a_w) of the treatment media, eventual advantages of UV light for pasteurization purposes will be higher in low a_w foods. E. coli STCC 4201 could be considered as a target when UV light processing of foods.     

Characterization of Escherichia coli from raw poultry in Belgium and impact on the detection of Campylobacter jejuni using Bolton broth

A comparative study examining Bolton broth and Preston broth for enrichment and reliable detection of Campylobacter jejuni (both healthy and freeze stressed cells) was performed. Tested as pure cultures, Bolton broth enabled faster resuscitation and growth of C. jejuni compared to Preston broth. When C. jejuni was co-incubated with extended-spectrum-beta-lactamase (ESBL) producing Escherichia coli isolated from Belgian poultry meat preparations, the latter dominated in the Bolton enrichment broth and crowded the mCCDA plates. This resulted in the inability to recover C. jejuni by ISO 10272-1:2006 standard method. Preston broth did not support the growth of the ESBL E. coli isolates, but showed longer detection time of C. jejuni compared to Bolton broth. The use of the same antibiotic (sodium cefoperazone) in Bolton broth and in mCCDA plates may explain the problems encountered for detection of C. jejuni, as high numbers of ESBL E. coli present after enrichment in Bolton broth, also caused overgrowth and masked the few C. jejuni colonies present on the mCCDA plates. The use of Campylobacter spp. specific real-time PCR circumvented these problems and enabled rapid detection of the pathogen after 24 h enrichment in both Bolton and Preston broth, for both healthy and freeze stressed cells.     

The prevalence of Listeria, Salmonella, Escherichia coli and E. coli O157:H7 on bison carcasses during processing

Bison meat is a relatively new, emerging meat species gaining increased popularity in the US and European meat markets, but little is known of its microflora or pathogens that may be present. This study was carried out to determine the incidence of the foodborne pathogens Listeria, Salmonella, Escherichia coli/E. coli O157:H7 on slaughtered bison and to evaluate the bison slaughter process. Bison carcass sampling was carried out at monthly intervals over a period of 1 year at a Bison processing facility in the Midwestern United States. A total of 355 Bison carcasses were sampled by surface swabbing the carcasses at five points on the production line: pre-dehiding, post-evisceration, post-USDA inspection, post-washing and 24 h chilled carcass. Overall, the prevalence of Listeria spp., Salmonella spp., E. coli and E. coli O157:H7 was 18.3%, 3.94%, 38.3% and 1.13%, respectively. The prevalence of Listeria spp. at each sampling point tested was 42.24%, 18.1%, 6.03%, 1.72% and 3.77% while the prevalence of E. coli at each sampling point was: 88.79%, 73.28%, 52.59%, 56.89% and 11.3%, respectively. The data obtained suggests that current antimicrobial intervention strategies used at the plant are relatively effective in reducing Listeria and E. coli contamination on bison carcasses to some extent, however further study is required to determine the influence of current slaughter practices on carcass contamination. The data reported in this study to the authors’ knowledge is some of the first information reporting on the bacteriological status of Bison, and provides some useful baseline information for future research.     

Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins

Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 μM isopropyl-β--thiogalactopyranoside (IPTG) at 37°C, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.     

Behavior of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium in teewurst, a raw spreadable sausage

The fate of Listeria monocytogenes, Salmonella Typhimurium, or Escherichia coli O157:H7 were separately monitored both in and on teewurst, a traditional raw and spreadable sausage of Germanic origin. Multi-strain cocktails of each pathogen (ca. 5.0 log CFU/g) were used to separately inoculate teewurst that was subsequently stored at 1.5, 4, 10, and 21 °C. When inoculated into commercially-prepared batter just prior to stuffing, in general, the higher the storage temperature, the greater the lethality. Depending on the storage temperature, pathogen levels in the batter decreased by 2.3 to 3.4, ca. 3.8, and 2.2 to 3.6 log CFU/g for E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively, during storage for 30 days. When inoculated onto both the top and bottom faces of sliced commercially-prepared finished product, the results for all four temperatures showed a decrease of 0.9 to 1.4, 1.4 to 1.8, and 2.2 to 3.0 log CFU/g for E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively, over the course of 21 days. With the possible exceptions for salt and carbohydrate levels, chemical analyses of teewurst purchased from five commercial manufacturers revealed only subtle differences in proximate composition for this product type. Our data establish that teewurst does not provide a favourable environment for the survival of E. coli O157:H7, S. Typhimurium, or L. monocytogenes inoculated either into or onto the product.     

Resistance of Listeria monocytogenes, Escherichia coli O157:H7 and Campylobacter jejuni after exposure to repetitive cycles of mild bactericidal treatments

While maintaining nutritional and sensorial attributes of fresh foods mild processing technologies generally deliver microbiologically perishable food products. Currently little information exists on possible increase in the resistance of pathogens after repetitive exposure to mild (sub-lethal) treatments. Multiple strain-cocktails of Listeria monocytogenes, Escherichia coli O157:H7 and Campylobacter jejuni were exposed to 20 consecutive cycles of sub-lethal inactivation by three different techniques. Used techniques comprised inactivation with lactic acid (LA), chlorine dioxide (ClO₂) and intense light pulses (ILP). Results showed that the selection of resistant cells was both species and technique dependent. While repetitive cycles of ClO₂ treatment did not result in increased resistance, repetitive inactivation with LA yielded L. monocytogenes culture of higher resistance in comparison to the parental culture. The increased resistance, expressed as decreased level of reduction in bacterial counts in subsequent inactivation cycles, was also observed with ILP for both L. monocytogenes and E. coli O157:H7 strains. Visual trend observations were confirmed through statistical linear regression analysis. No such effects were noted for C. jejuni which became undetectable after first 2–5 cycles. Current findings indicate the ability of foodborne pathogens to adapt to mild bactericidal treatments creating new challenges in risk assessment and more specifically in hazard analysis.     

Expression and purification of the recombinant mustard trypsin inhibitor 2 (MTI2) in Escherichia coli

The mustard trypsin inhibitor 2, MTI2, was expressed in Escherichia coli. A specific procedure for its production and purification is described. The recombinant protein was recovered by protein extraction from the insoluble fraction, then renatured and purified by ion exchange and gel filtration chromatography. Finally, the inhibitory activity against trypsin was also determined.     

Influence of organic acid concentration on survival of Listeria monocytogenes and Escherichia coli 0157:H7 in beef carcass wash water and on model equipment surfaces

Acid-adapted cultures of Escherichia coli O157:H7 and Listeria monocytogenes were inoculated in meat decontamination spray-washing runoff fluids in order to evaluate their survival and potential to form biofilms on stainless steel coupons. The cultures (10⁷ cfu ml⁻¹) and stainless steel coupons were exposed to mixtures of water and organic acid washings (composites of each of 2% acetic acid or lactic acid washings with water washings from meat decontamination in proportions of 1/9, 1/49, 1/99 [vol/vol]) or to water washings for up to 14 days at 15°C. E. coli O157:H7 formed biofilms and remained detectable (1.3 log cfu cm⁻²) on stainless steel for up to 4 d in the 1/9 dilution (pH 3.17–3.77) of the organic acid washings, and persisted throughout storage (14 d) in the 1/49 (pH 3.96–4.33) and 1/99 (pH 4.34–6.86) dilution of the organic acid washings. L. monocytogenes populations were unable to form detectable (<1.3 log cfu cm⁻²) biofilms in the 1/9 and 1/49 dilutions of both organic acid washings for up to 14 d; however, by day-14 in the 1/99 dilution of the washings, the pathogen was able to attach at detectable levels (2.7 to 3.4 logs). The pH effects of lower concentrations (1/49 or 1/99) of acidic washings decreased over time due to the formation of amine compounds produced by the natural meat flora, allowing resuscitation of the acid-stressed pathogen survivors. The resuscitation of acid-stressed pathogens may potentially enhance their survival and prevalence in biofilms and thus more attention should be focused on avoiding or minimizing the collection of decontamination runoff fluids on food contact equipment surfaces.     

Occurrence, virulence genes and antibiotic resistance of Escherichia coli O157 isolated from raw bovine, caprine and ovine milk in Greece

The examination of 2005 raw bovine (n = 950), caprine (n = 460) and ovine (n = 595) bulk milk samples collected throughout several regions in Greece for the presence of Escherichia coli serogroup O157 resulted in the isolation of 29 strains (1.4%) of which 21 were isolated from bovine (2.2%), 3 from caprine (0.7%) and 5 from ovine (0.8%) milk. Out of the 29 E. coli O157 isolates, only 12 (41.4%) could be classified as Shiga-toxigenic based on immunoassay and PCR results. All 12 Shiga-toxigenic E. coli serogroup O157 isolates belonged to the E. coli O157:H7 serotype. All except one of the 12 Shiga-toxin positive isolates were stx₂-positive, five of which were also stx₁-positive. The remaining isolate was positive only for the stx₁ gene. All stx-positive isolates (whether positive for stx₁, stx₂ or stx₁ and stx₂) were also PCR-positive for the eae and ehxA genes. The remaining 17 E. coli O157 isolates (58.6%) were negative for the presence of the H7 flagellar gene by PCR, tested negative for Shiga-toxin production both by immunoassay and PCR, and among these, only four and three strains were PCR-positive for the eae and ehxA genes, respectively. All 29 E. coli O157 isolates displayed resistance to a wide range of antimicrobials, with the stx-positive isolates being, on average, resistant to a higher number of antibiotics than those which were stx-negative.     

Effect of X-ray treatments on inoculated Escherichia coli O157: H7, Salmonella enterica, Shigella flexneri and Vibrio parahaemolyticus in ready-to-eat shrimp

This study was conducted to evaluate the inactivation effect of X-ray treatments on Escherichia coli O157: H7, Salmonella enteric (S. enterica), Shigella flexneri (S. flexneri) and Vibrio parahaemolyticus (V. parahaemolyticus) artificially inoculated in ready-to-eat (RTE) shrimp. A mixed culture of three strains of each tested pathogen was used to inoculate RTE shrimp. The shrimp samples were inoculated individually with selected pathogenic bacteria then aseptically placed in sterile plastic cups and air-dried at 22 °C for 30 min (to allow bacterial attachment) in the biosafety cabinet prior to X-ray treatments. The inoculated shrimp samples were then placed in sterilized bags and treated with 0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 2.0, 3.0 and 4.0 kGy X-ray at ambient temperature (22 °C and 60% relative humidity). Surviving bacterial populations were evaluated using a non-selective medium (TSA) with the appropriate selective medium overlay for each bacterium; CT-SMAC agar for E. coli O157: H7, XLD for S. enterica and S. flexneri and TCBS for V. parahaemolyticus. More than a 6 log CFU reduction of E. coli O157: H7, S. enterica, S. flexneri and V. parahaemolyticus was achieved with 2.0, 4.0, 3.0 and 3.0 kGy X-ray, respectively. Furthermore, treatment with 0.75 kGy X-ray significantly reduced the initial microflora on RTE shrimp samples from 3.8 ± 0.2 log CFU g⁻¹ to less than detectable limit (<1.0 log CFU g⁻¹).     

Molecular and epidemiological characterization of bovine intrauterine Escherichia coli

Escherichia coli are believed to be associated with postpartum metritis and endometritis but their role in the pathogenesis of both diseases is still undefined. In this study, uterine swabs for E. coli isolation were collected from 374 lactating Holstein cows housed on 4 commercial farms near Ithaca, New York. A total, 125 of 374 cows (33.4%) were positive for E. coli culture. Standard multiplex PCR protocols were used to screen the isolates for the presence of 32 virulence factor genes. Cows that had twin parturition were 4.4 times more likely to have intrauterine E. coli contamination than those that gave birth to single live female calves. Stillborn parturition and birth of single live male calves also increased the odds of intrauterine contamination by E. coli (3.7- and 1.6-fold, respectively) compared with birth of live female calves. Six virulence factors, common to extraintestinal and enteroaggregative E. coli, were found to be associated with metritis and endometritis: fimH, hlyA, cdt, kpsMII, ibeA, and astA. The virulence factor gene fimH was the most prevalent and the most significant: intrauterine E. coli carrying fimH and at least 1 of the other 5 identified virulence factors were pathogenic, and phylogenetic analysis based on the nucleotide sequence of DNA gyrase from 41 such IUEC revealed 2 clades.