Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

Detection of Escherichia coli O157:H7 with langasite pure shear horizontal surface acoustic wave sensors

The toxigenic Escherichia coli O157:H7 bacterium has been connected with hemorrhagic colitis and hemolytic uremic syndrome, which may be characterized by diarrhea, kidney failure and death. On average, O157:H7 causes 73,000 illnesses, 2100 hospitalizations and 60 deaths annually in the United States alone. There is the need for sensors capable of rapidly detecting dangerous microbes in food and water supplies to limit the exposure of human and animal populations. Previous work by the authors used shear horizontal surface acoustic wave (SH SAW) devices fabricated on langasite (LGS) Euler angles (0°, 22°, 90°) to successfully detect macromolecular protein assemblies. The devices also demonstrated favorable temperature stability, biocompatibility and low attenuation in liquid environments, suggesting their applicability to bacterial detection. In this paper, a biosensor test setup utilizing a small volume fluid injection system, stable temperature control and high frequency phase measurement was applied to validate LGS SH SAW biosensors for bacterial detection. The LGS SH SAW delay lines were fabricated and derivatized with a rabbit polyclonal IgG antibody, which selectively binds to E. coli O157:H7, in this case a non-toxigenic test strain. To quantify the effect of non-specific binding (negative control), an antibody directed against the trinitrophenyl hapten (TNP) was used as a binding layer. Test E. coli bacteria were cultured, fixed with formaldehyde, stained with cell-permeant nucleic acid stain, suspended in phosphate buffered saline and applied to the antibody-coated sensing surfaces. The biosensor transmission coefficient phase was monitored using a network analyzer. Phase responses of about 14° were measured for the E. coli detection, as compared to 2° due to non-specific anti-TNP binding. A 30:1 preference for E. coli binding to the anti-O157:H7 layer when compared to the anti-TNP layer was observed with fluorescence microscopy, thus confirming the selectivity of the antibody surface to E. coli.     

Cy5 labeled antimicrobial peptides for enhanced detection of Escherichia coli O157:H7

Fluorescently labeled antimicrobial peptides were evaluated as a potential replacement of labeled antibodies in a sandwich assay for the detection of Escherichia coli O157:H7. Antimicrobial peptides naturally bind to the lipopolysaccharide component of bacterial cell walls as part of their mode of action. Because of their small size relative to antibodies peptides can bind to cell surfaces with greater density, thereby increasing the optical signal and improving sensitivity. This method combines the specificity of a capture antibody with the increased sensitivity provided by using a labeled peptide as a detection molecule. The antimicrobial peptides cecropin P1, SMAP29, and PGQ were labeled with the fluorescent dye Cy5 via maleimide linker chemistry. Preliminary screening using a whole-cell solution binding assay revealed that Cy5 cecropin P1 enhanced the detection of E. coli O157:H7 relative to a Cy5 labeled anti-E. coli O157:H7 antibody 10-fold. Detection sensitivity of antibody and peptide were also compared with a prototype immuno-magnetic bead biosensor. Detection using Cy5 cecropin P1 resulted in a 10-fold improvement in sensitivity. Correlation of peptide antimicrobial activity with detection of E. coli O157:H7 indicated that activity was not predictive of the sensitivity of the fluorescent assay.     

The detection of Cryptosporidium parvum and Escherichia coli O157 in UK bivalve shellfish

Optimised immunomagnetic separation methods to detect Cryptosporidium parvum and Escherichia coli O157 in UK shellfish are described. Whole tissue homogenates gave the best recoveries for C. parvum oocysts compared with gill or haemolymph extracts. The sensitivity of recovery from spiked samples was comparable to that achieved when processing water and varied from 12–34% in mussels, 48–69.5% in oysters and 30–65% in scallops. Maximum recovery of E. coli O157 was achieved by enriching in buffered peptone water supplemented with vancomycin at 42 °C. Increasing enrichment temperatures from 37 to 42 °C gave a significant increase in target number recovery. Implementation of these methods into monitoring programmes and end-product testing will enable shellfish producers to better assess product safety.     

Effects of geometry on transmission and sensing potential of tapered fiber sensors

Geometry of tapered fiber sensors critically affects the response of an evanescent field sensor to cell suspensions. Single-mode fibers (nominally at 1300 nm) were tapered to symmetric or asymmetric tapers with diameters in the range of 3–20 μm, and overall lengths of 1–7 mm. Their transmission characteristics in air, water and in the presence of Escherichia coli (JM101 strain) at concentrations of 100, 1000, 7000 and 7 million cells/mL were measured in the 400–800 nm range and gave rich spectral data that lead to the following conclusions. (1) No change in transmission was observed due to E. coli with tapers that showed no relative change in transmission in water compared to air. (2) Tapers that exhibited a significant difference in transmission in water compared to air gave weak response to the presence of the E. coli. Of these, tapers with low waist diameters (6 μm) showed sensitivity to E. coli at 7000 cells/mL and higher concentration. (3) Tapers that showed modest difference in water transmission compared to air, and those that had small waist diameters gave excellent response to E. coli at 100–7000 cells/mL. In addition, mathematical modeling showed that: (1) at low wavelength (470 nm) and small waist diameter (6 μm), transmission with water in the waist region is higher than in air. (2) Small changes in waist diameter (0.05 μm) can cause larger changes in transmission at 470 nm than at 550 nm at waist diameter of 6 μm. (3) For the same overall geometry, a 5.5 μm diameter taper showed larger refractive index sensitivity compared to a 6.25 μm taper at 470 nm.     

Surface display of a glucose binding protein

Glucose binding protein (GBP) from Escherichia coli has been widely used to develop minimally invasive glucose biosensors for diabetics. To develop a cell-based glucose biosensor, it is essential to functionally display GBP on the cell surface. In this study, we designed a molecular structure to display GBP on the outer membrane of E. coli. We fused GBP with the first nine N-terminal residues of Lpp (major E. coli lipoprotein) and the 46–150 residues of OmpA (an outer membrane protein of E. coli). With this molecular design, we have successfully displayed GBP on the surface of E. coli. Using FITC-conjugated Dextran, we demonstrated that glucose’s binding sites of surface-displayed GBP were accessible to glucose. Furthermore, we showed that glucose transport in a GBP-deficient E. coli NM303 could be restored by displaying GBP on the surface of NM303. 0.51 h⁻¹ of specific growth rate was attained for NM303/pESDG grown in M9 minimal medium supplemented with 2 g/l glucose, whereas no growth was observed for NM303 in the same medium. Both NM303 and NM303/pESDG grew in M9 medium supplemented with 1 mM of fucose. Because cell surface is an interface between intracellular and extracellular molecular events, this technique paves a way to develop cell-based glucose biosensors.     

Verification of elicitor efficacy of lipopolysaccharides and peptidoglycans on antibacterial peptide gene expression in Bombyx mori

We verified the efficacy of lipopolysaccharide (LPS) in activating the cecropin B gene (CecB) in an immune-competent Bombyx mori cell line. Strong activation of CecB by the LPSs from Escherichia coli, Pseudomonas aeruginosa, and Salmonella minnesota were completely eliminated after digestion of the LPSs with muramidase. The results clearly indicate that a polymer form of PGN in the LPSs elicited CecB. An oligonucleotide microarray screen revealed that none of the 16,000 genes on the array were activated by LPS in the cells. In contrast, E. coli PGN strongly elicited five antibacterial peptide genes and numerous other genes, and PGN from Micrococcus luteus activated only several genes. Semi-quantitative RT-PCR revealed that all antibacterial genes activated by both PGNs, but the extents were 10–100 times higher with E. coli PGN. Similarly, higher elicitor activity of E. coli than M. luteus was indicated using peptidoglycan recognition protein gene, which is involved in pro-phenol oxidase cascade.     

A piezoelectric immunosensor for specific capture and enrichment of viable pathogens by quartz crystal microbalance sensor, followed by detection with antibody-functionalized gold nanoparticles

A sensitive bacteria enrichment and detection system for viable Escherichia coli O157:H7 was developed using a piezoelectric biosensor-quartz crystal microbalance (QCM) with antibody-functionalized gold nanoparticles (AuNPs) used as detection verifiers and amplifiers. In the circulating-flow QCM system, capture antibodies for E. coli O157:H7 were first immobilized onto the QCM chip. The sample containing E. coli O157:H7 was circulated through the system in the presence of 10ml of brain heart infusion (BHI) broth for 18h. The cells of E. coli O157:H7 specifically captured and enriched on the chip surface of the QCM were identified by QCM frequency changes. Listeria monocytogenes and Salmonella Typhimurium were used as negative controls. After bacterial enrichment, detection antibody-functionalized AuNPs were added to enhance the changes in detection signal. The use of BHI enrichment further enhanced the sensitivity of the developed system, achieving a detection limit of 0-1log CFU/ml or g. The real-time monitoring method for viable E. coli O157:H7 developed in this study can be used to enrich and detect viable cells simultaneously within 24h. The unique advantages of the system developed offer great potential in the microbial analysis of food samples in routine settings.     

Characterisation of E. coli O157 isolates from bovine hide and beef trimming in Irish abattoirs by pulsed field gel electrophoresis

Escherichia coli O157 isolates from bovine hide (n=117) and beef trimmings (n=32) from a single abattoir were examined by pulsed field gel electrophoresis (PFGE). Using BioNumerics software, dendrograms of isolates from each sample type (i.e. hide and beef trimming) were produced. In assessing the genetic relatedness of isolates, a similarity criterion of 80% was applied. The 117 E. coli O157 hide isolates were grouped into 14 clusters, comprising of 109 different PFGE profiles. Of the 109 different PFGE profiles, 8 were common to multiple isolates (i.e. shared 100% similarity by PFGE).

The 32 E. coli O157 beef trimming isolates produced 28 different PFGE profiles and 2 clusters. Of the 28 PFGE profiles, 2 were common to multiple isolates and the remaining 26 were distinct.

On a number of sampling occasions, isolates displaying identical PFGE patterns were recovered from multiple isolates collected from a single sample type (i.e. hides or trimmings), suggesting cross contamination from contaminated hides/animals to uncontaminated hides/animals and from contaminated beef trimmings to uncontaminated beef trimmings during abattoir operations.     

An over expression and high efficient mutation system of a cobalt-containing nitrile hydratase

A superior novel recombinant strain, E. coli BL21(DE3)/pETNH^M, containing the start codon mutation of the subunit, was constructed and selected as an overexpression and high efficient mutation platform for the genetic manipulation of the nitrile hydratase (NHase). Under optimal conditions, the specific activity of the recombinant strain reached as high as 452 U/mg dry cell. Enzymatic characteristics studies showed that the reaction activation energy of the recombinant NHase^M was 24.4 ± 0.5 kJ/mol, the suited pH range for catalysis was 5.5–7.5, and the K_m value was 4.34 g/L (82 mM). To assess the feasibility of the NHase improvement by protein rational design using this E. coli, site-directed mutagenesis of S122A, S122C, S122D and βW47E of the NHase^M were carried out. The NHase^M (S122A) and NHase^M (S122D) mutants were entirely inactive due to the charge change of the side-chain group. The product tolerance of the NHase^M (S122C) mutant was enhanced while its activity decreased by 30%. The thermo-stability of the NHase^M (βW47E) mutant was significantly strengthened, while its activity reduced by nearly 50%. These results confirmed that the specific activity of the mutant NHase expressed by the recombinant E. coli BL21(DE3)/pETNH^M can reasonably change with and without mutations. Therefore, this recombinant E. coli can be efficiently and confidently used for the further rational/random evolution of the NHase to simultaneously improve the activity, thermo-stability and product tolerance of the target NHase.     

An efficient cloning of DNA fragments by a method based on uracil-DNA glycosylase and endonuclease IV

We introduced a novel method to clone random DNA fragments independent of ligation reaction. The method involves the generation of long protruding ends on PCR amplification DNA. Both oligonucleotides used for the amplification of the vector DNA carried one uracil residue at the tenth position from the 5′ end and this made the creation of the 3′ protruding ends of linearized vector possible by uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV). 76 groups of annealed oligonucleotides that had ten-nucleotides protruding at 3′-end, which were complementary to those at 3′-end of the linearized vector, were designed. The linearized vector and the annealed oligonucleotide were mixed together to transform E.coli directly without ligation reaction. The number of the clone that grew on the plates had been demonstrated to reach 1 × 10⁵ transformants/μg and 96.1% of transformants harbored the cloned fragments. From the results of transformation, we can confirm that the efficiency of the creation of 3′ protruding ends in our method is high and our cloning method is benefit to produce recombinants easily and efficiently.     

Enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione: Cloning and expression of reductases

The enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione (1) to either of the corresponding (S)- and (R)-6-hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-diones (2 and 3, respectively) is described. The NADP⁺-dependent (R)-reductase (RHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (R)-6-hydroxybuspirone (3) was purified to homogeneity from cell extracts of Hansenula polymorpha SC 13845. The subunit molecular weight of the enzyme is 35,000 kDa based on sodium dodecyl sulfate gel electrophoresis and the molecular weight of the enzyme is 37,000 kDa as estimated by gel filtration chromatography. (R)-reductase from H. polymorpha was cloned and expressed in Escherichia coli. To regenerate the cofactor NADPH required for reduction we have cloned and expressed the glucose-6-phosphate dehydrogenase gene from Saccharomyces cerevisiae in E. coli. The NAD⁺-dependent (S)-reductase (SHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (S)-6-hydroxybuspirone (2) was purified to homogeneity from cell extracts of Pseudomonas putida SC 16269. The subunit molecular weight of the enzyme is 25,000 kDa based on sodium dodecyl sulfate gel electrophoresis. The (S)-reductase from P. putida was cloned and expressed in E. coli. To regenerate the cofactor NADH required for reduction we have cloned and expressed the formate dehydrogenase gene from Pichia pastoris in E. coli. Recombinant E. coli expressing (S)-reductase and (R)-reductase catalyzed the reduction of 1 to (S)-6-hyroxybuspirone (2) and (R)-6-hyroxybuspirone (3), respectively, in >98% yield and >99.9% e.e.     

Evaluation of 5'-nuclease and hybridization probe assays for the detection of shiga toxin-producing Escherichia coli in human stools

5′-Nuclease and a hybridization probe assays for the detection of shiga toxin-producing Escherichia coli were validated with regard to selectivity, analytical sensitivity, reproducibility and clinical performance. Both assays were capable of detecting the classical stx₁ and stx₂ genes when challenged with reference strains of E. coli (n = 40), although 1 to 4 minority sequence variants, whose clinical relevance is limited (stx_1c, stx_1d, and stx_2f), were detected less efficiently or not at all by one or both assays. No cross reaction was observed for both assays with 37 strains representing other gastrointestinal pathogens, or normal gastrointestinal flora. Analytical sensitivity ranged from 3.07 to 3.52 log₁₀ and 3.42 to 4.63 log₁₀ CFU/g of stool for 5′-nuclease and hybridization probe assay, respectively. Reproducibility was high with coefficients of variation of ≤ 5% for both inter- and intra-assay variation. Clinical performance was identical with a panel of archived positive specimens (n = 19) and a prospective panel of stools associated with bloody diarrhea (n = 115). In conclusion, both assays proved to be sensitive and reproducible.     

The structure of the neuraminic acid-containing capsular polysaccharide of Escherichia coli serotype K9

The acidic capsular polysaccharide isolated from Escherichia coli O9:K9:H12 was investigated by using n.m.r. spectroscopy, methylation analysis, periodate oxidation, and bacteriophage-borne enzyme degradation. The polysaccharide, the structure of which is shown below, is the third E. coli capsular polysaccharide reported to contain neuraminic acid, the others being the K1 and K92 polysaccharides, and it is the first in the E. coli series shown to contain a 4-linked neuraminic acid unit.     

Binding of MutS protein to oligonucleotides containing a methylated or an ethylated guanine residue, and correlation with mutation frequency

The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O⁶-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O⁶-MeG:T, O⁶-MeG:C, O⁶-EtG:T, O⁶-EtG:C, G:T and G:C. Dissociation constants (K_d), which reflect the strength of binding, followed the order G:T- > O⁶-MeG:T- > O⁶-EtG:T- = O⁶-EtG:C- ≥ O⁶-MeG:C- > G:C. These results suggest that a thymine base paired with O⁶-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O⁶-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.     

Bactericidal Activity of Catecholamine Copper Complexes

Washed or growing E. coli cells are killed by epinephrine, norepinephrine or dopamine in the presence of non lethal concentrations of Cu(II). Killing is enhanced by anoxia and by sublethal Concentrations of H₂O₁. The rate of killing is proportional to the rate of catecholamine oxidation. The copper epinephrine complex binds to E. coli cells, induces membrane damage and depletion of the cellular ATP pool. The cells may be partially protected by SOD or catalase but not by OH radical scavengers. Addition of H²O₂ to cells which were sensitized by preincubation with the epinephrine-copper complex, causes rapid killing and DNA degradation. Sensitized cells are not protected by BSA.     

A MEMS based amperometric detector for E. coli bacteria using self-assembled monolayers.

We developed a system for amperometric detection of Escherichia coli (E. coli) based on the integration of microelectromechanical systems (MEMS), self-assembled monolayers (SAMS), DNA hybridization, and enzyme amplification. Using MEMS technology, a detector array was fabricated which has multiple electrodes deposited on a Si wafer and was fully reusable. Using SAMs, a monolayer of the protein streptavidin was immobilized on the working electrode (Au) surface to capture rRNA from E. coli. Three different approaches can be used to immobilize streptavidin onto Au, direct adsorption of the protein on bare Au, binding the protein to a biotinylated thiol SAM on Au, and binding the protein to a biotinylated disulfide monolayer on Au. The biotinylated thiol approach yielded the best results. High specificity for E. coli was achieved using ssDNA–rRNA hybridization and high sensitivity was achieved using enzymatic amplification with peroxidase as the enzyme. The analysis protocol can be conducted with solution volumes on the order of a few microliters and completed in 40 min. The detection system was capable of detecting 1000 E. coli cells without polymerase chain reaction with high specificity for E. coli vs. the bacteria Bordetella bronchiseptica.     

Methionine analogues as inhibitors of methionyl-tRNA synthetase

A series of methionine analogues have been synthesized as inhibitors of methionyl-tRNA synthetase and evaluated for their inhibitory activities of E. coli methionyl-tRNA synthetase and bacterial growth. Among them, -methionine hydroxamate 20 has proved to be the best inhibitor of the enzyme with K_i = 19 μM and showed a growth inhibition against E.coli JM 109, P. vulganis 6059 and C. freundii 8090.