Carbon microarrays for the direct impedimetric detection of Bacillus anthracis using Gamma phages as probes.

A direct and efficient impedimetric method is presented for the detection of Bacillus anthracis Sterne vegetative cells, using Gamma phages as probes attached to screen-printed carbon electrode microarrays. The carbon electrodes were initially functionalized through cyclic-voltammetric reduction of a nitro-aryl diazonium moiety, followed by further reduction of nitro groups to amino groups, and finally by treatment with glutaraldehyde. Functionalization (probe immobilization) using Gamma phages was verified by XPS and TOF-SIM experiments. The Gamma phage-modified microarrays were then used to detect B. anthracis Sterne bacteria in aqueous electrolyte media. Faradaic impedimetric detection of bacteria in KCl solution containing the ferri/ferro cyanide redox couple shows a gradual increase in Z' (real impedance) values, taken from the extrapolation of the linear portion of Nyquist plots in the low frequency range, for sensors placed in contact with increasing concentrations of B. anthracis. ΔZ' values vary from 700 to 5300 Ohms for bacteria concentrations ranging from 10(2) to 10(8) cfu mL(-1). These shifts in Z' are attributed to a decrease in diffusion controlled charge transfer to the electrode surface following capture of intact B. anthracis. No significant ΔZ' was observed for control experiments using E. coli. K12 as a non-specific target, even at a concentration of 10(8) cfu mL(-1).

Bacteriophage-modified microarrays for the direct impedimetric detection of bacteria.

A novel method is presented for the specific and direct detection of bacteria using bacteriophages as recognition receptors immobilized covalently onto functionalized screen-printed carbon electrode (SPE) microarrays. The SPE networks were functionalized through electrochemical oxidation in acidic media of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) by applying a potential of +2.2 V to the working electrode. Immobilization of T4 bacteriophage onto the SPEs was achieved via EDC by formation of amide bonds between the protein coating of the phage and the electrochemically generated carboxylic groups at the carbon surface. The surface functionalization with EDC, and the binding of phages, was verified by time-of-flight secondary ion mass spectrometry. The immobilized T4 phages were then used to specifically detect E. coli bacteria. The presence of surface-bound bacteria was verified by scanning electron and fluorescence microscopies. Impedance measurements (Nyquist plots) show shifts of the order of 10(4) Omega due to the binding of E. coli bacteria to the T4 phages. No significant change in impedance was observed for control experiments using immobilized T4 phage in the presence of Salmonella. Impedance variations as a function of incubation time show a maximum shift after 20 min, indicating onset of lysis, as also confirmed by fluorescence microscopy. Concentration-response curves yield a detection limit of 10(4) cfu/mL for 50-microL samples.

Recent advances in research on radiofrequency fields and health: 2001-2003.

The widespread use of wireless telecommunications devices, particularly mobile phones, has resulted in increased human exposure to radiofrequency (RF) fields. Although national and international agencies have established safety guidelines for exposure to RF fields, concerns remain about the potential for adverse health outcomes to occur in relation to RF field exposure. The extensive literature on RF fields and health has been reviewed by a number of authorities, including the Royal Society of Canada (1999), the European Commission's Scientific Committee on Toxicity, Ecotoxicity, and the Environment (CSTEE, 2001), the British Medical Association (2001), the Swedish Radiation Protection Authority (Boice & McLaughlin, 2002), and the Health Council of The Netherlands (2002). This report provides an update on recent research results on the potential health risks of RF fields since the publication of the Royal Society of Canada report in 1999 (See Krewski et al., 2001a) and our previous 2001 update (Krewski et al., 2001b), covering the period 2001-2003. The present report examines new data on dosimetry and exposure assessment, biological effects such as enzyme induction, and toxicological effects, including genotoxicity, carcinogenicity, and testicular and reproductive outcomes. Epidemiological studies of mobile phone users and occupationally exposed populations are examined, along with human and animal studies of neurological and behavioral effects. All of the authoritative reviews completed within the last 2 yr have concluded that there is no clear evidence of adverse health effects associated with RF fields. However, following a recent review of nine epidemiological studies of mobile phones and cancer, Kundi et al. (2004) concluded that the possibility of an enhanced cancer risk cannot be excluded. These same reviews support the need for further research to clarify the possible associations between RF fields and adverse health outcomes that have appeared in some reports. The results of the ongoing World Health Organization (WHO) study of mobile phones will provide important new information in this regard.

Detection of multiple antibiotic-resistant Salmonella enterica serovar Typhimurium DT104 by phage replication-competitive enzyme-linked immunosorbent assay.

A phage replication-competitive enzyme-linked immunosorbent assay (PR-cELISA) was developed for the detection of multiple antibiotic-resistant Salmonella Typhimurium DT104. In the PR-cELISA procedure, a phage, BP1, was inoculated into a log-phase bacterial culture at a ratio of 1:100. After a 3-h incubation of the mixture, BP1 replication was measured by cELISA based on the competitive binding between BP1 and biotinylated BP1 to Salmonella Typhimurium smooth lipopolysaccharide. Among the 84 Salmonella strains and 9 non-Salmonella strains that were tested by PR-cELISA, BP1 detected 39 of 40 Salmonella Typhimurium strains, 2 of 10 Salmonella non-Typhimurium somatic group B strains, and 5 of 18 Salmonella somatic group D1 strains. With the addition of chloramphenicol to the culture medium, PR-cELISA detected all 27 multiple antibiotic-resistant Salmonella Typhimurium DT104 and none of the other Salmonella strains or non-Salmonella strains tested. The results demonstrated that PR-cELISA has potential applications for the detection of multiple antibiotic-resistant Salmonella Typhimurium DT104.

Immune markers and ornithine decarboxylase activity among electric utility workers.

The effects of a 60-Hz magnetic field (MF) exposure on white blood cell ornithine decarboxylase (ODC) activity, natural killer (NK) cell activity, lymphocyte phenotypes, and differential cell counts were studied among 60 electric utility workers. Personal MF exposure monitoring over 3 consecutive workdays was followed by collection of a peripheral blood sample. There were no MF-related changes in NK activity or the number of circulating neutrophils, eosinophils, basophils, or T-lymphocytes (CD4, CD8, CD4:CD8 ratio). MF exposure intensity was associated with decreased ODC activity (P<0.01) and lower NK cell counts (P=0.04). Melatonin production, which stimulates the immune system, was quantified on the night preceding immune marker determinations. Exposure-related reductions in ODC activity, NK and B cells, and monocytes were strongest among workers with reduced melatonin production. The biological significance or long-term health consequences associated with these changes are not known.

Anthrax-protective effects of yeast beta 1,3 glucans.

CONTEXT: The recent events increasing the threat of bioterrorism have prompted a widespread search for defenses against this peril. OBJECTIVE: To evaluate the anthrax-protective effect of beta1,3-glucan immune modulators (PGG-glucan and WGP beta glucan) in an experimental animal model. DESIGN: Beta1,3-glucan immune modulators were administered by subcutaneous injection to Balb/c mice 2 days prior to anthrax challenge. WGP beta glucan was administered by daily oral gavage for 7 days prior to challenge, or in drinking water for 10 days postchallenge with a lethal dose of Bacillus anthracis spores. Survival, survival time, and microbial bioburden relative to an infected, untreated control group were assessed. RESULTS: A single injected dose of PGG-glucan or WGP beta glucan immune modulators given 2 days before challenge significantly: (a) increased the survival rate of infected mice (2.5-fold), (b) diminished the bacterial load in the lungs of infected mice (4-8-fold), and (c) increased the proportion of bacteria-free animals 10 days after challenge (2-fold). In mice prophylactically administered oral WGP beta glucan for 1 week prior to infection, survival increased from 50% to 100%; therapeutic administration of oral WGP beta glucan for 10 days postinfection increased survival from 30% up to 90% in treatment groups. CONCLUSIONS: These results demonstrate the potential for beta1,3-glucan immune modulators to provide a significant degree of protection against anthrax, a potential biological warfare (BW) agent in a mouse model of anthrax infection. Further studies are needed to optimize protection, evaluate activity in combination with other treatment options, demonstrate activity in a validated primate model of infection, and determine if protection is effective against other potential BW agents.

Magnetically-assisted impedimetric detection of bacteria using phage-modified carbon microarrays.

This study presents an investigation on the possibility of improving the detection limit of bacteria with an inexpensive electrochemical, impedimetric sensor platform, by integrating the sensor with magnetic manipulation. The approach uses T4 bacteriophage coated Dynabeads to selectively capture and concentrate E. coli K12 cells from samples, to increase the sensitivity of detection at the surface of functionalized screen-printed carbon microarrays. Fluorescence and flow cytometry measurements indicate that the surface modification of the magnetic beads, with phages, and binding with the bacteria, were successful. Integration of the screen-printed carbon-based impedimetric sensor, with a magnetic manipulation system, was found to improve the sensitivity of the device, decreasing the limit of detection of E. coli K12 from 10(4) to 10(3) cfu/mL. We have also demonstrated that this approach provides for more specific detection of bacteria, enabling the operator to account for non-specific adsorption, and detection of bacteria in more complex (real) samples (milk).

The differential detection of methicillin-resistant, methicillin-susceptible and borderline oxacillin-resistant Staphylococcus aureus by surface plasmon resonance.

Two hundred fifty Staphylococcus aureus clinical isolates were studied to determine their susceptibilities to ß-lactam antibiotics. Among these isolates, 16 were methicillin-sensitive S. aureus (MSSA), 207 were methicillin-resistant S. aureus (MRSA) and 27 were borderline oxacillin-resistant S. aureus (BORSA). Currently, the reported mechanism of methicillin resistance in S. aureus is the production of a distinctive penicillin binding protein 2a (PBP2a), which exhibits low affinity toward ß-lactams. A surface plasmon resonance biosensor was evaluated for its ability to identify MRSA and to distinguish these strains from MSSA and BORSA, by specifically detecting PBP2a. We found that the system permits label-free, real-time, specific detection of pathogens for concentrations as low as 10 colony forming units/milliliter (CFU/ml), in less than 20 min. This system promises to become a diagnostic tool for bacteria that cause major public concern in clinical settings.

Surface plasmon resonance detection of E. coli and methicillin-resistant S. aureus using bacteriophages.

Early diagnosis and appropriate treatment of Escherichia coli (E. coli) O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) are key elements in preventing resultant life-threatening illnesses, such as hemorrhagic colitis, hemolytic uremic syndrome, and septicemia. In this report, we describe the use of surface plasmon resonance (SPR) for the biodetection of pathogenic bacteria, using bacteriophages as the recognition elements. T4 bacteriophages were used to detect E. coli, while a novel, highly specific phage was used to detect MRSA. We found that the system permits label-free, real-time, specific, rapid and cost-effective detection of pathogens, for concentrations of 10(3) colony forming units/milliliter, in less than 20 min. This system promises to become a diagnostic tool for bacteria that cause major public concern for food safety, bioterrorism, and nosocomial infections.