Isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 from ground beef using modified rainbow agar and post-immunomagnetic separation acid treatment.

It is estimated that at least 70% of human illnesses due to non-O157 Shiga toxin-producing Escherichia coli (STEC) in the United States are caused by strains from the top six serogroups (O26, O45, O103, O111, O121, and O145). Procedures for isolating STEC from food products often use plating media that include antimicrobial supplements at concentrations that inhibit background microflora growth but can also inhibit target STEC growth. In this study, an agar medium with lower supplement concentrations, modified Rainbow agar (mRBA), was evaluated for recovery of STEC serogroups O26, O45, O103, O111, O121, and O145 from ground beef enrichments. A post-immunomagnetic separation (IMS) acid treatment step was additionally used to reduce background microflora and increase recovery of target STEC strains. Ground beef samples (325 g) were artificially contaminated with STEC and confounding organisms and enriched for 15 h. Recovery of the target STEC was attempted on the enrichments using IMS and plating onto mRBA and Rainbow agar (RBA). Additionally, acid treatment was performed on the post-IMS eluate followed by plating onto mRBA. Using the combination of mRBA and acid treatment, target STEC were isolated from 103 (85.8%) of 120 of the low-inoculated samples (1 to 5 CFU/325-g sample) compared with 68 (56.7%) of 120 using no acid treatment and plating onto RBA with higher levels of novobiocin and potassium tellurite. The combination of acid treatment and mRBA provides a significant improvement over the use of RBA for isolation of STEC serogroups O26, O45, O103, O111, O121, and O145 from raw ground beef.

Prescreening bacterial colonies for bioactive molecules with Janus plates, a SBS standard double-faced microbial culturing system.

Despite the availability of many culture-based antibiotic screening methods, the lack of sensitive automated methods to identify functional molecules directly from microbial cells still limits the search for new biologically active compounds. The effectiveness of antibiotic detection is influenced by the solubility of the assayed compounds, indicator strain sensitivity, culture media and assay configuration. We describe a qualitative high throughput screening system for detecting cell-perturbing molecules from bacterial colonies employing two opposed agar layers sequentially formed in prototype Society for Biomolecular Screening (SBS) plates, named Janus plates. Direct assay of microbial colonies against target organisms in opposed agar layers overcomes some of the limitations of agar overlay methods. The system enables the rapid detection of extracellular cell-perturbing molecules, e.g., antibiotics, excreted directly from environmental isolates. The source bacterial colonies remain separate from the target organism. The growth layer is prepared and grown independently, so environmental strains can be grown for longer intervals, at temperatures and in media that favor their growth and metabolite expression, while the assay layer with pathogens, usually requiring nutrient-rich medium and elevated temperatures, are added later. Colonies to be tested can be precisely arrayed on the first agar surface, thus avoiding dispersion and disturbance of potential antibiotic-producing colonies by overlaying agar with the target strain. The rectangular SBS configuration facilitates factorial replication of dense microbial colony arrays for testing with multiple assays and assay conditions employing robotic colony pickers and pin tools. Opposed agar layers only slightly reduced the effectiveness for detecting growth inhibition from pure antibiotics compared to single-layer agar diffusion assays. The Janus plate enabled an automation-assisted workflow where a lone operator can effectively identify and accumulate bioactive soil bacterial strains within a few weeks. We also envisage the method's utility for functional prescreening colonies of clones from genomic and metagenomic libraries or improved strains originating from mutagenized cells.

STD sensor based on nucleic acid functionalized nanostructured polyaniline.

STD (sexually transmitted disease, Gonorrhoea) sensor based on nucleic acid probe (from Opa, a multi-copy gene of Neisseria gonorrhoeae) functionalized nanostructured-polyaniline coated onto indium-tin-oxide-coated glass plate has been fabricated using avidin-biotin as cross-linking agent. This DNA functionalized electrode can specifically detect upto 0.5 x 10(-15)M of complementary target within 60s of hybridization time at 25 degrees C by differential pulse voltammetry (DPV) using methylene blue as electro-active DNA hybridization indicator. This highly sensitive and specific nucleic acid functionalized nanostructured-polyaniline electrode can distinguish presence of N. gonorrhoeae from Neisseria meningitidis and Escherichia coli culture and spiked samples from the urethral swabs of the patients.

Monitoring of Mycoplasma genitalium growth and evaluation of antibacterial activity of antibiotics tetracycline and levofloxacin using a wireless magnetoelastic sensor.

Mycoplasma genitalium (Mg) is the smallest and simplest self-replicating bacteria lacking of cell wall and is a human pathogen causing various diseases. This paper describes the real-time, long-term and in situ monitoring of the growth of Mg and evaluation of the effect of the antibiotics tetracycline and levofloxacin on the growth using a wireless magnetoelastic sensor. The sensor is fabricated by coating a magnetoelastic strip with a polyurethane protecting film. In response to a time-varying magnetic field, the sensor longitudinally vibrates at a resonance frequency, emitting magnetic flux that can be remotely detected by a pick-up coil. No physical connections between the sensor and the detection system are required. The wireless property facilitates aseptic operation. The adhesion of Mg on the sensor surface results in a decrease in the resonance frequency, which is proportional to the concentration of Mg. The shift of the resonance frequency-time curves shows that under routine culture condition the growth curve of Mg is composed of three phases those are lag, logarithmic and stationary phase, respectively. In the presence of the antibiotics, the lag phase in the growth inhibition curves is prolonged obviously and the stationary phase is substituted by a decline phase. The growth inhibition of Mg is related to the concentration of the antibiotics. The MIC50 (minimal inhibitory concentration) of Mg incubated in the presence of the antibiotics for 120h is calculated to be 1.5 and 0.5 microg/mL for tetracycline and levofloxacin, respectively.

A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157:H7.

A quartz crystal microbalance (QCM) DNA sensor, based on the nanoparticle amplification method, was developed for detection of Escherichia coli O157:H7. A thiolated single-stranded DNA (ssDNA) probe specific to E. coli O157:H7 eaeA gene was immobilized onto the QCM sensor surface through self-assembly. The hybridization was induced by exposing the ssDNA probe to the complementary target DNA, and resulted in the mass change and therefore frequency change of the QCM. Streptavidin conjugated Fe(3)O(4) nanoparticles (average diameter=145 nm) were used as "mass enhancers" to amplify the frequency change. Synthesized biotinylated oligonucleotides as well as E. coli O157:H7 eaeA gene fragments (151 bases) amplified using asymmetric PCR with biotin labeled primers were tested. As low as 10(-12)M synthesized oligonucleotides and 2.67 x 10(2) colony forming unit (CFU)/ml E. coli O157:H7 cells can be detected by the sensor. Linear correlation between frequency change and logarithmic number of bacterial cell concentration was found for E. coli O157:H7 from 2.67 x 10(2) to 2.67 x 10(6)CFU/ml.

A microsystem compatible strategy for viable Escherichia coli detection.

This study delineates a microsystem compatible strategy that enables the rapid determination of Escherichia coli viability for the application in food and water monitoring. This approach differentiates the living cells from the dead ones by detecting the presence of a "viability indicator", i.e. mRNAs of a common E. coli GroEL heat shock protein (hsp). Our method starts with a stimulated and controlled transcription of hsp mRNA under an elevated temperature (47 degrees C) for 20min. Following that, the short-life mRNA is rapidly extracted using streptavidin-modified magnetic particles containing biotin-labeled DNA probes complementary to a specific region of the mRNA. The quantification of mRNA by gel electrophoresis and Ag/Au-based electrochemical detection is done after the amplification of mRNAs by reverse transcription-polymerase chain reaction (RT-PCR). Heat shock temperatures and durations that have profound effect to the mRNA transcription were studied and it was found that the mRNA undergoes a rapid minute-by-minute self-degradation after the environment resumes room temperature. Issues such as the DNA contamination that interfere the magnetic particle-based mRNA extraction technique were tackled. A sensitive Ag/Au-based electrochemical analysis method was used to detect the RT-PCR products and a cell concentration as low as 10(2)cfu/ml can be achieved by the electrochemical method, but not by the conventional gel electrophoresis. The strategy demonstrated in this study can be readily implemented in a microsystem and is a step forward for the realization of an integrated bioanalytical microsystem (lab on a chip) for the viable cell detection.