Polysaccharide-based silver nanoparticles synthesized by <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> DSM 29614 cause DNA fragmentation in <Emphasis Type="Italic">E. coli</Emphasis> cells

Silver nanoparticles (AgNPs), embedded into a specific exopolysaccharide (EPS), were produced by Klebsiella oxytoca DSM 29614 by adding AgNO₃ to the cultures during exponential growth phase. In particular, under aerobic or anaerobic conditions, two types of silver nanoparticles, named AgNPs-EPS^aer and the AgNPs-EPS^anaer, were produced respectively. The effects on bacterial cells was demonstrated by using Escherichia coli K12 and Kocuria rhizophila ATCC 9341 (ex Micrococcus luteus) as Gram-negative and Gram-positive tester strains, respectively. The best antimicrobial activity was observed for AgNPs-EPS^aer, in terms of minimum inhibitory concentrations and minimum bactericidal concentrations. Observations by transmission electron microscopy showed that the cell morphology of both tester strains changed during the exposition to AgNPs-EPS^aer. In particular, an electron-dense wrapped filament was observed in E. coli cytoplasm after 3 h of AgNPs-EPS^aer exposition, apparently due to silver accumulation in DNA, and both E. coli and K. rhizophila cells were lysed after 18 h of exposure to AgNPs-EPS^aer. The DNA breakage in E. coli cells was confirmed by the comparison of 3-D fluorescence spectra fingerprints of DNA. Finally the accumulation of silver on DNA of E. coli was confirmed directly by a significant Ag⁺ release from DNA, using the scanning electrochemical microscopy and the voltammetric determinations.     

An Efficient Catalytic DNA that Cleaves L-RNA

Many DNAzymes have been isolated from synthetic DNA pools to cleave natural RNA (D-RNA) substrates and some have been utilized for the design of aptazyme biosensors for bioanalytical applications. Even though these biosensors perform well in simple sample matrices, they do not function effectively in complex biological samples due to ubiquitous RNases that can efficiently cleave D-RNA substrates. To overcome this issue, we set out to develop DNAzymes that cleave L-RNA, the enantiomer of D-RNA, which is known to be completely resistant to RNases. Through in vitro selection we isolated three L-RNA-cleaving DNAzymes from a random-sequence DNA pool. The most active DNAzyme exhibits a catalytic rate constant ~3 min^-1 and has a structure that contains a kissing loop, a structural motif that has never been observed with D-RNA-cleaving DNAzymes. Furthermore we have used this DNAzyme and a well-known ATP-binding DNA aptamer to construct an aptazyme sensor and demonstrated that this biosensor can achieve ATP detection in biological samples that contain RNases. The current work lays the foundation for exploring RNA-cleaving DNAzymes for engineering biosensors that are compatible with complex biological samples.     

Development of SCAR primers based on a repetitive DNA fingerprint for Escherichia coli detection

The present study aimed to use enterobacterial repetitive intergenic consensus (ERIC) fingerprints to design SCAR primers for the detection of Escherichia coli. The E. coli strains were isolated from various water sources. The primary presumptive identification of E. coli was achieved using MacConkey agar. Nineteen isolates were selected and confirmed to be E. coli strains based on seven biochemical characteristics. ERIC-PCR with ERIC 1R and ERIC 2 primers were used to generate DNA fingerprints. ERIC-PCR DNA profiles showed variant DNA profiles among the tested E. coli strains and distinguished all E. coli strains from the other tested bacterial strains. A 350 bp band that predominated in five E. coli strains was used for the development of the species-specific SCAR primers EC-F1 and EC-R1. The primers showed good specificity for E. coli, with the exception of a single false positive reaction with Sh. flexneri DMST 4423. The primers were able to detect 50 pg and 10⁰ CFU/ml of genomic DNA and cells of E. coli, respectively.     

Direct Inhibition of <Emphasis Type="Italic">Col</Emphasis> E<Subscript>1</Subscript> Plasmid DNA Replication in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Rifampicin

COLICINOGENIC factor E₁ (Col E₁) is a small bacterial plasmid (4.2×10⁶ daltons) present in colicinogenic strains of Escherichia coli¹ to the extent of about twenty-four copies per cell (Clewell and Helinski, unpublished results), which continues to replicate in the presence of high levels of chloramphenicol, a specific inhibitor of protein synthesis, although the chromosome only completes current rounds of replication and ceases (Clewell and Helinski, unpublished results). The average rate of Col E₁ semiconservative replication in the absence of protein synthesis is, in certain conditions, faster than (as much as eight times) the normal rate of synthesis (Clewell, unpublished results). Replication continues for 10–15 h after the addition of chloramphenicol, resulting in nearly 3,000 copies of Col E¹ DNA per cell. We are taking advantage of this system to study the effects of a number of antibiotics on DNA replication and now report evidence that rifampicin (an active semisynthetic derivative of rifamycin B)², an antibiotic known specifically to inhibit bacterial DNA dependent RNA polymerase^3–6, has a dramatic inhibitory effect on Col E¹ DNA replication.     

Comparative study of HOCl-inflicted damage to bacterial DNA ex vivo and within cells

The prospects for using bacterial DNA as an intrinsic probe for HOCl and secondary oxidants/chlorinating agents associated with it has been evaluated using both in vitro and in vivo studies. Single-strand and double-strand breaks occurred in bare plasmid DNA that had been exposed to high levels of HOCl, although these reactions were very inefficient compared to polynucleotide chain cleavage caused by the OH-generating reagent, peroxynitrite. Plasmid nicking was not increased when intact Escherichia coli were exposed to HOCl; rather, the amount of recoverable plasmid diminished in a dose-dependent manner. At concentration levels of HOCl exceeding lethal doses, genomic bacterial DNA underwent extensive fragmentation and the amount of precipitable DNA-protein complexes increased several-fold. The 5-chlorocytosine content of plasmid and genomic DNA isolated from HOCl-exposed E. coli was also slightly elevated above controls, as measured by mass spectrometry of the deaminated product, 5-chlorouracil. However, the yields were not dose-dependent over the bactericidal concentration range. Genomic DNA recovered from E. coli that had been subjected to phagocytosis by human neutrophils occasionally showed small increases in 5-chlorocytosine content when compared to analogous cellular reactions where myeloperoxidase activity was inhibited by azide ion. Overall, the amount of isolable 5-chlorouracil from the HOCl-exposed bacterial cells was far less than the damage manifested in polynucleotide bond cleavage and cross-linking.     

Multiplex PCR and DNA array for the detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. targeting virulence-related genes

A multiplex PCR and DNA array for quick detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. was developed using specific genetic markers derived from virulence-related genes. The genetic markers of cytK, sei, prfA, rfB, and hilA gene specifically amplified DNA fragments of 320 bp, 500 bp, 700 bp, 1.0 kb and 1.2 kb from B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., respectively. These markers are specific for the detection of the corresponding target pathogens. The sensitivity of the genetic markers was down to ～0.5 fg genomic DNA and ～10¹ CFU/ml (one bacterial cell per reaction) of bacterial culture. The combination of mPCR and DNA macroarray hybridization sensitively and specifically detected B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., in complex mixed cultures and food matrices. Thus, this mPCR and macroarray-based approach serves as rapid and reliable diagnostic tool for the detection of these five pathogens.     

<Emphasis Type="Italic">E. coli</Emphasis> DNA Polymerase I and other Host Functions participate in T4 DNA Replication and Recombination

The recognition of bacterial functions involved in DNA metabolism of bacteriophage T4 might reveal interactions between different enzymes during DNA replication and recombination. To detect such functions we have studied the replication of complete and incomplete T4 chromosomes in various mutant strains of Escherichia coli that are defective in their own DNA metabolism. We found that several E. coli functions can substitute for phage functions in T4 replication and recombination and will discuss here the role of the E. coli pol A gene which codes for DNA polymerase I^1–4 and of the dna B and E genes^3,5.     

Transforming DNA Uptake Gene Orthologs Do Not Mediate Spontaneous Plasmid Transformation in Escherichia coli

Spontaneous plasmid transformation of Escherichia coli occurs on nutrient-containing agar plates. E. coli has also been reported to use double-stranded DNA (dsDNA) as a carbon source. The mechanism(s) of entry of exogenous dsDNA that allows plasmid establishment or the use of DNA as a nutrient remain(s) unknown. To further characterize plasmid transformation, we first documented the stimulation of transformation by agar and agarose. We provide evidence that stimulation is not due to agar contributing a supplement of Ca²⁺, Fe²⁺, Mg²⁺, Mn²⁺, or Zn²⁺. Second, we undertook to inactivate the E. coli orthologues of Haemophilus influenzae components of the transformation machine that allows the uptake of single-stranded DNA (ssDNA) from exogenous dsDNA. The putative outer membrane channel protein (HofQ), transformation pseudopilus component (PpdD), and transmembrane pore (YcaI) are not required for plasmid transformation. We conclude that plasmid DNA does not enter E. coli cells as ssDNA. The finding that purified plasmid monomers transform E. coli with single-hit kinetics supports this conclusion; it establishes that a unique monomer molecule is sufficient to give rise to a transformant, which is not consistent with the reconstitution of an intact replicon through annealing of partially overlapping complementary ssDNA, taken up from two independent monomers. We therefore propose that plasmid transformation involves internalization of intact dsDNA molecules. Our data together, with previous reports that HofQ is required for the use of dsDNA as a carbon source, suggest the existence of two routes for DNA entry, at least across the outer membrane of E. coli.The spontaneous transformation of Escherichia coli with plasmid DNA on nutrient-containing agar plates was described in at least three independent articles (14, 23, 24). However, no attempt to characterize the mechanism of plasmid DNA uptake has been reported. Genomic analysis revealed the presence in E. coli of a set of genes homologous to those required for DNA uptake in naturally transformable species, including the gram-positive Bacillus subtilis and Streptococcus pneumoniae and the gram-negative Haemophilus influenzae and Neisseria gonorrhoeae (9). The machine they potentially encode would allow the uptake of single-stranded DNA (ssDNA) from an exogenous double-stranded DNA (dsDNA) substrate in E. coli (Fig. ​(Fig.1).1). HofQ (called ComE in reference 7) is the ortholog of the PilQ secretin of N. gonorrhoeae, which constitutes a transmembrane channel required for exogenous dsDNA to traverse the outer membrane (OM) and reach the so-called transformation pseudopilus (8). According to the Bacillus subtilis paradigm (8), assembly of the pseudopilus requires a prepilin peptidase (PppA; called PilD in reference 7), a traffic NTPase (HofB; called PilB in reference 7), and a polytopic membrane protein (HofC; called PilC in reference 7). The pseudopilus, which would include PpdD (called PilA in reference 7), provides access for dsDNA to its receptor, YbaV (called ComE1 in reference 7), through the peptidoglycan. Degradation of one strand by an unidentified nuclease (N) would allow uptake of ssDNA through YcaI (called Rec2 in reference 7), a channel in the inner membrane. Finally, DprA (also named Smf) would be required to protect internalized ssDNA from endogenous nucleases, as shown in S. pneumoniae (4), and to assist the processing of ssDNA into transformants (16).Open in a separate windowFIG. 1.Diagrammatic representation of the putative E. coli DNA uptake machine. The E. coli orthologues of proteins required involved in the uptake of transforming DNA in naturally transformable species, including B. subtilis, S. pneumoniae, H. influenzae, and N. gonorrhoeae, were identified by genomic analysis (9). GspD is a PilQ paralogue (25% identity over 278 residues), which was considered in the present study as a possible alternative route for dsDNA across the OM. A prepilin peptidase (PppA; called PilD in reference 7) required for maturation and export of proteins constituting the transformation pseudopilus (see Table S1 in the supplemental material) is not drawn on this diagram. (Additional information regarding the relationship between E. coli and H. influenzae transformation genes, and a table listing the various alternative names used in the literature are available in the supplemental material.). Red crosses indicate components of the putative DNA uptake machine inactivated during this work. IM, inner membrane.In H. influenzae, transformation genes are preceded by unusual CRP (for cyclic AMP receptor protein) binding sites, now called CRP-S (7), that absolutely require a second protein, Sxy (also called TfoX), in addition to CRP for induction (19). Interestingly, bioinformatics analysis revealed the conservation of CRP-S sites in front of the corresponding E. coli genes (7), including all of the genes encoding the proteins shown in Fig. ​Fig.11 (except GspD). Furthermore, some of these genes were experimentally demonstrated to require CRP, cAMP (CRP''s allosteric effector), and Sxy for induction in E. coli, providing support to the view that CRP-S sites control a bona fide transformation regulon in this bacterium (7). However, the involvement of E. coli transformation genes in DNA uptake has not been documented, except for hofQ, which was reported to be required for the use of dsDNA as a nutrient (11, 18). Although the functionality of the E. coli transformation genes has not been confirmed experimentally, it is of note that the bioinformatics identification of a complete set of transformation genes in two other species not previously known to be naturally transformable, Streptococcus thermophilus and Bacillus cereus, opened the way to the demonstration of genetic transformation in these species (6, 15a).To characterize further spontaneous plasmid transformation in E. coli, we first identified parameters affecting plasmid transformation frequencies on plates. We then undertook to inactivate genes encoding the putative transformation-related DNA uptake machinery of E. coli (Fig. ​(Fig.1)1) and to compare the rate of spontaneous plasmid transformation in the corresponding mutants and in their wild-type parent. In addition, to get an insight into the process of plasmid DNA entry, we characterized the kinetics of plasmid monomer transformation because it was shown in S. pneumoniae that regeneration of an intact plasmid replicon requires the independent uptake (via the transformation machine) of complementary ssDNA from two monomers (21). Finally, we discuss the possible significance of our data regarding the entry of exogenous dsDNA in E. coli in the light of previous findings on the use of dsDNA as a carbon source in this species (11, 18).     

Enzyme-linked lectinosorbent assay of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In this study, we developed a microplate sandwich analysis of Escherichia coli and Staphylococcus aureus bacterial pathogens based on the interaction of their cell wall carbohydrates with natural receptors called lectins. An immobilized lectin-cell-biotinylated lectin complex was formed in this assay. Here, we studied the binding specificity of several plant lectins to E. coli and S. aureus cells, and pairs characterized by high-affinity interactions were selected for the assay. Wheat germ agglutinin and Ricinus communis agglutinin were used to develop enzyme-linked lectinosorbent assays for E. coli and S. aureus cells with the detection limits of 4 × 10⁶ and 5 × 10⁵ cells/mL, respectively. Comparison of the enzyme-linked immonosorbent assay and the enzyme-linked lectinosorbent assay demonstrated no significant differences in detection limit values for E. coli. Due to the accessibility and universality of lectin reagents, the proposed approach is a promising tool for the control of a wide range of bacterial pathogens.     

A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine-Pyrimidine Junction

Several RNA-cleaving deoxyribozymes (DNAzymes) have been reported for efficient cleavage of purine-containing junctions, but none is able to efficiently cleave pyrimidine-pyrimidine (Pyr-Pyr) junctions. We hypothesize that a stronger Pyr-Pyr cleavage activity requires larger DNAzymes with complex structures that are difficult to isolate directly from a DNA library; one possible way to obtain such DNAzymes is to optimize DNA sequences with weak activities. To test this, we carried out an in vitro selection study to derive DNAzymes capable of cleaving an rC-T junction in a chimeric DNA/RNA substrate from DNA libraries constructed through chemical mutagenesis of five previous DNAzymes with a k_obs of ∼ 0.001 min^− 1 for the rC-T junction. After several rounds of selective amplification, DNAzyme descendants with a k_obs of ∼ 0.1 min^− 1 were obtained from a DNAzyme pool. The most efficient motif, denoted “CT10-3.29,” was found to have a catalytic core of ∼ 50 nt, larger than other known RNA-cleaving DNAzymes, and its secondary structure contains five short duplexes confined by a four-way junction. Several variants of CT10-3.29 exhibit a k_obs of 0.3-1.4 min^− 1 against the rC-T junction. CT10-3.29 also shows strong activity (k_obs  > 0.1 min^− 1) for rU-A and rU-T junctions, medium activity (> 0.01 min^− 1) for rC-A and rA-T junctions, and weak activity (> 0.001 min^− 1) for rA-A, rG-T, and rG-A junctions. Interestingly, a single-point mutation within the catalytic core of CT10-3.29 altered the pattern of junction specificity with a significantly decreased ability to cleave rC-T and rC-A junctions and a substantially increased ability to cleave rA-A, rA-T, rG-A, rG-T, rU-A, and rU-T junctions. This observation illustrates the intricacy and plasticity of this RNA-cleaving DNAzyme in dinucleotide junction selectivity. The current study shows that it is feasible to derive efficient DNAzymes for a difficult chemical task and reveals that DNAzymes require more complex structural solutions for such a task.     

DNA condensation in bacteria: Interplay between macromolecular crowding and nucleoid proteins

The volume of a typical Eschericia coli nucleoid is roughly 10⁴ times smaller than the volume of a freely coiling linear DNA molecule with the same length as the E. coli genome. We review the main forces that have been suggested to contribute to this compaction factor: macromolecular crowding (that “pushes” the DNA together), DNA charge neutralization by various polycationic species (that “glues” the DNA together), and finally, DNA deformations due to DNA supercoiling and nucleoid proteins. The direct contributions of DNA supercoiling and nucleoid proteins to the total compaction factor are probably small. Instead, we argue that the formation of the bacterial nucleoid can be described as a consequence of the influence of macromolecular crowding on thick, supercoiled protein-DNA fibers, that have been partly charge neutralized by small multivalent cations.     

Multiplex real-time PCR assay for detection of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and screening for non-O157 Shiga toxin-producing <Emphasis Type="Italic">E. coli</Emphasis>

Background

Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency.

Methods

The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s.

Results

The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella enterica, Shigella strains, or any other pathogenic strains tested.

Conclusions

A multiplex real-time PCR assay that can rapidly and simultaneously detect E. coli O157:H7 and screen for non-O157 STEC strains has been developed and assessed for efficacy. The inclusivity and exclusivity tests demonstrated high sensitivity and specificity of the multiplex real-time PCR assay. In addition, this multiplex assay was shown to be effective for the detection of E. coli O157:H7 from two common food matrices, beef and spinach, and may be applied for detection of E. coli O157:H7 and screening for non-O157 STEC strains from other food matrices as well.

     

Change in the Template Specificity of RNA Phage SP-Replicase by the <Emphasis Type="Italic">E. coli</Emphasis> Cell Component

Qβ-REPLICASE was isolated from E. coli infected with the RNA bacteriophage Qβ as RNA-dependent RNA polymerase which had template specificity¹. RNA phage SP², which is distinct from RNA phages isolated previously^3,4, has been isolated in our laboratory and SP-replicase⁵ was purified from E. coli infected with SP-phage. SP-replicase has a template specificity different from that of Qβ-replicase. By using this new RNA-replicase, comparison between two distinct replicases has become possible.     

Lyophilization Prior to Direct DNA Extraction from Bovine Feces Improves the Quantification of Escherichia coli O157:H7 and Campylobacter jejuni

Lyophilization was used to concentrate bovine feces prior to DNA extraction and analysis using real-time PCR. Lyophilization significantly improved the sensitivity of detection compared to that in fresh feces and was associated with reliable quantification of both Escherichia coli O157:H7 and Campylobacter jejuni bacteria present in feces at concentrations ranging between 2 log₁₀ and 6 log₁₀ CFU g⁻¹.Bovines are a reservoir for verotoxigenic Escherichia coli O157:H7 and Campylobacter jejuni, pathogenic microorganisms responsible for severe human gastrointestinal disease (5, 12). Qualitative and quantitative detection of these organisms in bovine feces is essential for evaluating risk to human health. Real-time PCR (quantitative PCR [qPCR]) assays have been developed to detect and quantify both E. coli O157:H7 and C. jejuni bacteria by using DNA directly extracted from animal feces (20, 22). Analysis of DNA extracted from bovine feces can generate a high level of correlation between the actual target cell density and the PCR signal (7, 8). However, the detection of E. coli O157:H7 and C. jejuni by direct DNA extraction is less sensitive and more variable than detection by procedures based on a preliminary enrichment step (e.g., laboratory culture) (7, 9, 16, 20). We explored the potential of lyophilization for improving overall detection by qPCR through increasing the amount of bovine fecal material available for DNA extraction.Four sets of five fresh bovine fecal samples were collected, and each sample was divided into four equal portions. Samples were seeded with either (i) E. coli O157:H7 (strain NZRM 3614) grown for 18 h at 37°C in tryptic soy broth (BD, Sparks, MD) or (ii) C. jejuni (strain NZRM 1958) grown for 48 h at 42°C in Exeter broth (11) to obtain the following concentrations: set 1, 0 CFU g⁻¹ (unseeded) and 3.5 log₁₀, 4.5 log₁₀, and 5.5 log₁₀ CFU of E. coli O157:H7 g⁻¹, and set 2, 0 CFU g⁻¹ (unseeded) to 5.2 log₁₀ CFU of E. coli O157:H7 g⁻¹. Set 3 and 4 concentrations varied from 0 CFU g⁻¹ (unseeded) to 6.4 log₁₀ C. jejuni CFU g⁻¹. DNA was either extracted directly from fresh samples or extracted from samples after lyophilization. Lyophilization involved mixing of prepared fecal samples in phosphate-buffered saline (145 mM NaCl, 59 mM Na₂HPO₄, 8 mM KH₂PO₄, pH 7.5) at a ratio of 1:10 (wt/vol), homogenization with a lab blender model 400 (Seward Medical, London, United Kingdom), cooling to −35°C, and concentration using a 1015GP lyophilizer (Cuddon Ltd., Blenheim, New Zealand). Total DNA was extracted from 0.2 g of a fresh or lyophilized fecal sample by using a QIAamp DNA stool minikit (Qiagen Inc., Mississauga, Canada). DNA was amplified using either a TaqMan E. coli O157:H7 detection kit (Applied Biosystems, Foster City, CA) or mapA primers and a corresponding probe (1). Amplification and fluorescence data were collected with optical-grade 96-well plates by using a TaqMan 7300 PCR system (Applied Biosystems). For each DNA sample, a mean threshold cycle (C_T) value for triplicate qPCR runs was calculated. When no C_T value was obtained, an arbitrary C_T value of 40 was assigned. All data were reported as equivalent concentrations in fresh feces. Significance levels were determined by one-way analysis of variance. The relationship between the log₁₀ numbers of CFU g⁻¹ fresh feces (viable-cell counts) and C_T values was analyzed using GenStat software (version 10.2.0.175; VSN International, Oxford, United Kingdom). Confidence intervals were obtained using the software program Flexi (21).Lyophilized samples were associated with significantly improved sensitivity (P < 0.001) at seeding levels of 4.5 and 5.5 log₁₀ E. coli O157:H7 CFU g⁻¹ (Table ​(Table1).1). At 3.5 log₁₀ CFU g⁻¹, the rate of E. coli O157:H7 detection was also higher, with all lyophilized samples producing a C_T value of <40 (Table ​(Table1).1). Individual C_T values for the three qPCR amplification runs were sufficiently similar to allow averaging (P > 0.05). Regression analysis of the averaged set 2 and 3 data (Fig. ​(Fig.1)1) demonstrated that the detection of both E. coli O157:H7 and C. jejuni was linear for seeding levels ranging from ca. 2 log₁₀ to 6 log₁₀ CFU g⁻¹ fresh feces. The range of concentrations used reflects the reported range of concentrations of these bacteria in feces (i.e., 0 to 6 log₁₀ CFU g⁻¹) as determined by conventional culture (3, 4, 18, 19). The high coefficients of correlation for the relationships between the log₁₀ numbers of CFU g⁻¹ feces and the C_T values indicated the specific amplification of the target DNA. The reproducibility of detection of E. coli O157:H7 was reduced at the lowest seeding concentration (i.e., 2.2 log₁₀ CFU g⁻¹ feces), with 75% of the samples giving a C_T value of <40. The limit for 100% successful detection after lyophilization was 2.9 log₁₀ E. coli O157:H7 CFU g⁻¹. The detection of C. jejuni by qPCR varied between sets. For set 3, 100% reproducibility occurred at 2.2 log₁₀ C. jejuni CFU g⁻¹. For set 4, satisfactory detection was obtained only after dilution of the DNA extract prior to qPCR. Despite this requirement for dilution, C. jejuni was still detected in 80% of the samples of set 4 seeded at a density of 2.2 log₁₀ C. jejuni CFU g⁻¹.Open in a separate windowFIG. 1.Ranges of quantification of E. coli O157:H7 (A) and C. jejuni (B) bacteria obtained from lyophilized fecal samples by real-time PCR. Each point represents the average C_T value for triplicate runs of one fecal sample at one seeding concentration. The hatched areas represent the 95% confidence intervals.

TABLE 1.

Difference in C_T values obtained for real-time PCR detection of E. coli O157:H7 in seeded fecal samples (n = 5) with and without lyophilization

DNA Microarray-Based Identification of Serogroups and Virulence Gene Patterns of Escherichia coli Isolates Associated with Porcine Postweaning Diarrhea and Edema Disease

Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 10³ CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated E. coli in porcine feces samples.     

Single Walled Carbon Nanotube-Based Junction Biosensor for Detection of Escherichia coli

Foodborne pathogen detection using biomolecules and nanomaterials may lead to platforms for rapid and simple electronic biosensing. Integration of single walled carbon nanotubes (SWCNTs) and immobilized antibodies into a disposable bio-nano combinatorial junction sensor was fabricated for detection of Escherichia coli K-12. Gold tungsten wires (50 µm diameter) coated with polyethylenimine (PEI) and SWCNTs were aligned to form a crossbar junction, which was functionalized with streptavidin and biotinylated antibodies to allow for enhanced specificity towards targeted microbes. In this study, changes in electrical current (ΔI) after bioaffinity reactions between bacterial cells (E. coli K-12) and antibodies on the SWCNT surface were monitored to evaluate the sensor''s performance. The averaged ΔI increased from 33.13 nA to 290.9 nA with the presence of SWCNTs in a 10⁸ CFU/mL concentration of E. coli, thus showing an improvement in sensing magnitude. Electrical current measurements demonstrated a linear relationship (R² = 0.973) between the changes in current and concentrations of bacterial suspension in range of 10²–10⁵ CFU/mL. Current decreased as cell concentrations increased, due to increased bacterial resistance on the bio-nano modified surface. The detection limit of the developed sensor was 10² CFU/mL with a detection time of less than 5 min with nanotubes. Therefore, the fabricated disposable junction biosensor with a functionalized SWCNT platform shows potential for high-performance biosensing and application as a detection device for foodborne pathogens.     

Molecular Basis for Repressor Activity of Qβ Replicase

WITH the purification and characterization of viral replicases, a novel feature of nucleic acid polymerases—stringent template specificity—was recognized^1,2. Qβ replicase, the most extensively studied viral RNA polymerase^2–8, is now known to replicate Qβ RNA², the complementary Qβ minus strand⁹, RNA molecules described as “variants” of Qβ RNA^10,11 and a set of small RNAs of unknown origin which accumulate in Qβ-infected Escherichia coli, collectively designated as “6S RNA”¹². On the other hand, the RNA from phages related distantly, if at all, to Qβ^13,14, such as MS2 or R17 and of other viruses such as TMV² or AMV (Diggelmann and Weissmann, unpublished results) are completely inert as templates, as are ribosomal and tRNA from E. coli². Poly C and C-rich synthetic copolymers at high concentrations elicit synthesis which, however, remains restricted to the formation of a strand complementary to the template^15,16.     

The effect of UV irradiation on the capacity of an Hfr recA strain of Escherichia coli to act as donor

Summary The ability of a recA Hfr strain of Escherichia coli to form colonies is extremely sensitive to inhibition by ultraviolet light (Fig. 2). Furthermore, in this strain the synthesis of DNA is stopped completely by a dose of 385 ergs/mm² of UV (Fig. 3). Nevertheless, the ability of this recA Hfr strain to act as a donor in sexual recombination was no more sensitive to UV than that of a wild type donor (Fig. 1). Furthermore, when irradiated and mated with a recA female, in which DNA synthesis was also inhibited by UV (Fig. 3), there was a net synthesis of DNA as measured by the incorporation of C¹⁴ thymidine (Fig. 4). By using nalidixic acid resistant recA donors and recipients in all combinations, irradiating with UV and treating with nalidixic acid during mating, it is shown that DNA was synthesized by the donor (Fig. 5). It is concluded that synthesis of DNA directed by the sex factor during mating in a recA donor is not as sensitive to inhibition by UV as normal DNA synthesis in a recA donor.