Synthesis and antimicrobial activities of structurally novel S,S'-bis(heterosubstituted) disulfides.

The central focus of this study is on the antibacterial and antifungal properties of synthetically produced S,S'-bis(heterosubstituted) disulfides as a means to control the growth of various infection-causing pathogens. Staphylococcus aureus, Francisella tularensis and Candida albicans were each found to be highly susceptible to several of these compounds by agar or broth dilution and Kirby-Bauer diffusion assays. These structurally simple, low molecular weight disulfides have shown promising bioactivities and may serve as leads to the development of effective new antibacterials for pathogenic bacteria such as methicillin-resistant S. aureus and F. tularensis.

Hollow-fiber ultrafiltration and PCR detection of human-associated genetic markers from various types of surface water in Florida.

Hollow-fiber ultrafiltration (HFUF) and PCR were combined to detect human-associated microbial source tracking marker genes in large volumes of fresh and estuarine Florida water. HFUF allowed marker detection when membrane filtration did not, demonstrating HFUF's ability to facilitate detection of diluted targets by PCR in a variety of water types.

Same-day detection of Escherichia coli O157:H7 from spinach by using electrochemiluminescent and cytometric bead array biosensors.

Contamination of fresh produce with Escherichia coli O157:H7 and other pathogens commonly causes food-borne illness and disease outbreaks. Thus, screening for pathogens is warranted, but improved testing procedures are needed to allow reproducible same-day detection of low initial contamination levels on perishable foods, and methods for detecting numerous pathogens in a single test are desired. Experimental procedures were developed to enable rapid screening of spinach for E. coli O157:H7 by using multiplex-capable immunological assays that are analyzed using biosensors. Detection was achieved using an automated electrochemiluminescent (ECL) assay system and a fluorescence-based cytometric bead array. Using the ECL system, less than 0.1 CFU of E. coli O157:H7 per gram of spinach was detected after 5 h of enrichment, corresponding to 6.5 h of total assay time. Using the cytometric bead array, less than 0.1 CFU/g was detected after 7 h of enrichment, with a total time to detection of less than 10 h. These results illustrate that both biosensor assays are useful for rapid detection of E. coli O157:H7 on produce in time frames that are comparable to or better than those of other testing formats. Both methods may be useful for multiplexed pathogen detection in the food industry and other testing situations.

Sensitive detection of multiplex toxins using antibody microarray.

Using a newly developed fluorescent nanoparticle (NP) that gives rise to a high-intensity and stable fluorescent light, a sensitive antibody (Ab) microarray assay system has been developed for specific detection of bioterrorism agents, as exemplified by ricin, cholera toxin (CT), and staphylococcal enterotoxin B (SEB). The Ab microarray uses a sandwich format that consists of capture Abs, analytes (toxins), biotinylated detection Abs, and avidin-conjugated NP. In all three cases, polyclonal Abs (pAbs) displayed superiority over monoclonal antibodies (mAbs) in capturing toxins on microarray slides even when the pAbs and mAbs had similar affinity as determined by enzyme-linked immunosorbent assay (ELISA). The detection system was successfully used to detect toxins spiked in milk, apple cider, and blood samples. We were able to detect ricin at 100 pg/ml in buffer and at 1 ng/ml in spiked apple cider or milk, whereas CT and SEB were detected at 10 pg/ml in buffer and 100 pg/ml in spiked apple cider or milk. High specificities were also demonstrated in the detection of mixed toxin samples with similar sensitivities. The matrix effect of blood samples on the detection of mixed toxins seems to be minimal when the toxin concentration is at or above 100 ng/ml. The current study highlights the significant role of pAb and NP in increasing selectivity and sensitivity of toxin detection in a microarray format.

Rapid detection and identification of bacterial pathogens by using an ATP bioluminescence immunoassay.

Rapid identification of viable bacterial contaminants in food products is important because of their potential to cause disease. This study examined a method for microbial detection by using a combined ATP bioluminescence immunoassay. Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium were selected as target organisms because of their implication in foodborne illness. Various matrices containing the target cells were examined, including ground beef homogenate, apple juice, milk, and phosphate-buffered saline. Specific antibodies were immobilized on the surface of 96-well plates, and then the sample matrices containing target cells in the wells were incubated. Sample matrix (no cells) was used to establish background. The plates were washed, and the wells were incubated with BacTiter-Glo reagent in Mueller-Hinton II broth. Bioluminescent output was measured with the GloMax 96 luminometer. Signal-to-noise ratios were calculated, resulting in a limit of detection of 10(4) CFU/ml for both E. coli O157:H7 and Salmonella Typhimurium. The limit of detection for both species was not affected by the presence of nontarget cells. The various sample matrices did not affect signal-to-noise ratios when E. coli O157:H7 was the target. A weak matrix effect was observed when Salmonella Typhimurium was the target. A strong linear correlation was observed between the number of cells and luminescent output over 4 orders of magnitude for both species. This method provides a means of simultaneously detecting and identifying viable pathogens in complex matrices, and could have wider application in food microbiology.

Rapid dead-end ultrafiltration concentration and biosensor detection of enterococci from beach waters of Southern California.

Same-day microbial water quality assessments are not possible with standard methods, which increases the possibility of public exposure to fecal pathogens. This study examined the efficacy of high-volume hollow fibre ultrafiltration coupled to biosensor detection for enterococci in marine waters to allow same-day public notification of poor water quality. Fifty-six 1001 ultrafiltered samples and 100 ml grab samples were collected weekly from May to July 2007. Post-ultrafiltration processing included sonication and micron sieve passage to remove interfering particulates, followed by centrifugation for secondary concentration. Levels of enterococci in grab and ultrafiltration samples were determined by a standard method (EPA method 1600) for calculation of recovery efficiencies and concentration factors. Each final retentate was analysed with the RAPTOR evanescent wave biosensor. Enterococci levels increased over 26,000-fold in final retentates. Enterococci were detected when ambient concentrations exceeded the regulatory standard for a single sample (> or = 105 CFU/100 ml), and detection was highly correlated with breaches of the single-sample regulatory limit. The combined procedure required 2.5 h for detection compared with 24h for EPA method 1600. This field study achieved rapid detection of enterococci by ultrafiltration, secondary concentration and biosensor analysis, and demonstrates its potential usefulness for water quality monitoring.

Physical properties and biological activity of poly(butyl acrylate-styrene) nanoparticle emulsions prepared with conventional and polymerizable surfactants.

Recent efforts in our laboratory have explored the use of polyacrylate nanoparticles in aqueous media as stable emulsions for potential applications in treating drug-resistant bacterial infections. These emulsions are made by emulsion polymerization of acrylated antibiotic compounds in a mixture of butyl acrylate and styrene (7:3 wt/wt) using sodium dodecyl sulfate as a surfactant. Prior work in our group established that the emulsions required purification to remove toxicity associated with extraneous surfactant present in the media. This article summarizes our investigations of poly(butyl acrylate-styrene) emulsions made using anionic, cationic, zwitterionic, and noncharged (amphiphilic) surfactants, as well as attachable surfactant monomers (surfmers), comparing the cytotoxicity and microbiological activity levels of the emulsion both before and after purification. Our results show that the attachment of a polymerizable surfmer onto the matrix of the nanoparticle neither improves nor diminishes cytotoxic or antibacterial effects of the emulsion, whether or not the emulsions are purified, and that the optimal properties are associated with the use of the nonionic surfactants versus those carrying anionic, cationic, or zwitterionic charge. Incorporation of an N-thiolated beta-lactam antibacterial agent onto the nanoparticle matrix via covalent attachment endows the emulsion with antibiotic properties against pathogenic bacteria such as methicillin-resistant Staphylococcus aureus, without changing the physical properties of the nanoparticles or their emulsions. FROM THE CLINICAL EDITOR: Emulsions of polyacrylate nanoparticles, antibiotics and surfactants were studied using surfactant monomers as controls. Nonionic surfactants resulted in the most optimal properties. Incorporation of a beta-lactam antibacterial agent onto the nanoparticle matrix endowed the emulsion with antibiotic properties against methicillin-resistant Staphylococcus aureus (MRSA), a leading cause of hospital acquired, treatment-resistant infections including sepsis.

Rapid ultrafiltration concentration and biosensor detection of enterococci from large volumes of Florida recreational water.

Monitoring recreational waters for fecal contamination by standard methodologies involves culturing indicator bacteria, such as fecal coliforms and enterococci. Delayed reporting of microbial water quality parameters increases the likelihood of public exposure to pathogens of fecal origin, making the development of rapid methods important for public health protection. A rapid assay for enterococci was developed using a combined ultrafiltration-biosensor procedure. Twelve 100-liter water samples were collected from upper Tampa Bay over a 9-month period. The samples were collected on site by dead-end hollow-fiber ultrafiltration. Postfiltration processing of the initial retentates included sonication and micrometer-level sieve passage to remove interfering particles. Centrifugation was utilized for secondary concentration. Grab samples were collected simultaneously with the ultrafiltered samples. Concentrations of enterococci in all grab and ultrafiltration samples were determined by the standard method (EPA method 1600) for calculation of recovery efficiencies and concentration factors. Levels of enterococci increased twofold in initial retentates and by 4 orders of magnitude in final retentates over ambient concentrations. An aliquot of each final retentate was adsorbed onto polystyrene waveguides for immunoassay analysis of enterococci with a microfluidic fiber optic biosensor, the Raptor. Enterococci were detected when concentrations in the ambient water exceeded the regulatory standard for a single sample (> or =105 CFU/100 ml). The combined ultrafiltration-biosensor procedure required 2.5 h for detection compared to 24 for the standard method. This study demonstrated that enterococci can be detected rapidly using on-site ultrafiltration, secondary concentration, and biosensor analysis.

Monitoring biosensor capture efficiencies: development of a model using GFP-expressing Escherichia coli O157:H7.

One of the known limitations for biosensor assays is the high limit of detection for target cells within complex samples (e.g., Escherichia coli at 10(4) to 10(5) CFU/mL) due to poor capture efficiencies. Currently, researchers can only estimate the cell capture efficiency necessary to produce a positive signal for any type of biosensor using either cumbersome techniques or regression modeling. To solve this problem, green fluorescent protein (GFP) transformed E. coli O157:H7 was used to develop a novel method for directly and easily measuring the cell capture efficiency of any given biosensor platform. For demonstration purposes, E. coli-GFP was assayed on both fiber optic and planar waveguide biosensor platforms. Cells were enumerated using an epifluorescent microscope and digital camera to determine the number of cells captured on the surfaces. Conversion algorithms were used with these digital images to determine the cell density of entire waveguide surface areas. For E. coli-GFP, the range of cell capture efficiency was between 0.4 and 1.2%. This indicates that although the developed model works for calculating cell capture, there is still need for significant improvements in capture methods themselves, to increase the capture efficiency and thereby lower detection limits. The use of GFP-transformed target cells and cell capture efficiency calculations can facilitate the development and optimization processes by allowing direct enumeration of new biosensor design configurations and sample processing strategies.

Glyconanobiotics: Novel carbohydrated nanoparticle antibiotics for MRSA and Bacillus anthracis.

This report describes the synthesis and evaluation of glycosylated polyacrylate nanoparticles that have covalently-bound antibiotics within their framework. The requisite glycosylated drug monomers were prepared from one of three known antibiotics, an N-sec-butylthio beta-lactam, ciprofloxacin, and a penicillin, by acylation with 3-O-acryloyl-1,2-O-isopropylidene-5,6 bis((chlorosuccinyl)oxy)-d-glucofuranose (7) or 6-O-acetyl-3-O-acryloyl-1,2-O-isopropylidene-5-(chlorosuccinyl)oxy-alpha-d-glucofuranose (10). These acrylated monomers were subjected to emulsion polymerization in a 7:3 (w:w) mixture of butyl acrylate-styrene in the presence of sodium dodecyl sulfate as surfactant (3 weight %) and potassium persulfate as a radical initiator (1 weight %). The resulting nanoparticle emulsions were characterized by dynamic light scattering and found to have similar diameters ( approximately 40 nm) and size distributions to those of our previously studied systems. Microbiological testing showed that the N-sec-butylthio beta-lactam and ciprofloxacin nanoparticles both have powerful in vitro activities against methicillin-resistant Staphylococcus aureus and Bacillus anthracis, while the penicillin-bound nanoparticles have no antimicrobial activity. This indicates the need for matching a suitable antibiotic with the nanoparticle carrier. Overall, the study shows that even relatively large, polar acrylate monomers (MW>1000 amu) can be efficiently incorporated into the nanoparticle matrix by emulsion polymerization, providing opportunities for further advances in nanomedicine.

Studies on the antifungal properties of N-thiolated beta-lactams.

N-thiolated beta-lactams had previously been shown to have antibacterial activity against a narrow selection of pathogenic bacteria including Staphylococcus aureus and Bacillus anthracis, as well as apoptotic-inducing activity in a variety of human cancer cell lines. We now have found that these lactams also possess antifungal activity against Candida and other fungi by exerting powerful cytostatic effects that disrupt the structural integrity of cytoplasmic membranes. The mode of action and structure-activity trends of these lactams as antifungals parallel that previously seen in our antibacterial studies.

Evanescent-wave biosensor for field serodiagnosis of tortoise mycoplasmosis.

Disease has become an increasingly important issue for wildlife management over the past two decades. Adequate surveillance is fundamental for disease prevention and control, thus there is an increasing need for diagnostic assays for wildlife management. The objective of this study was to evaluate the performance of a field-portable biosensor adapted for rapid detection of specific antibodies in tortoise plasma that reflect a history of exposure to Mycoplasma agassizii, which is an agent of tortoise upper respiratory tract disease. Banked plasma samples were tested in two blinded trials, and the parameters that define the reliability of a diagnostic test were estimated based on externally validated tortoise plasma controls. The mean sensitivity of the biosensor (ability to identify exposed tortoises in the group of all exposed individuals) was 78%; the mean specificity (unexposed individuals with negative test result, out of all unexposed individuals tested) was 73%; the mean positive predictive value (exposed individuals with positive test, out of all individuals with positive test) was 82%; the mean negative predictive value (unexposed individuals with negative test, out of all individuals with negative test) was 68%. In a 15-min field-portable format, the biosensor was able to discriminate between true seropositive (n=34) and true seronegative (n=23) tortoise plasma with overall accuracy of 84%. The goals established for the tortoise population can help managers decide whether potential diagnostic errors should impact management decision-making, and whether the benefits of the field-portable format of the biosensor assay outweigh any potential disadvantages.

Rapid PCR confirmation of E. coli O157:H7 after evanescent wave fiber optic biosensor detection.

Escherichia coli O157:H7 is an enteric pathogen of public health importance, which is monitored by several government agencies. Many rapid detection tests have been developed to identify foodstuff and water supplies contaminated by E. coli O157:H7. However, these methods can be time consuming (24-48 h) due to the need to culture the bacteria to confirm detection results. Fiber optic biosensors can rapidly detect pathogens from complex matrices, yet confirmation tests can take up to 10h to complete. In addition, fiber optic biosensors can also be used to reduce the impact of PCR inhibitors present in complex matrices such as food and water. This paper presents methodologies to reduce the time necessary for confirmation from 10 to about 2 h, by direct PCR of bacteria from the fiber optic waveguides without the need for culture or enrichment steps.

A rapid detection method for Vaccinia virus, the surrogate for smallpox virus.

Prior to the World Health Organization's announcement of total eradication in 1977 [J. Am. Med. Assoc. 281 (1999) 1735], smallpox was a worldwide pathogen. Vaccinations were ceased in 1980 and now with a largely unprotected world population, smallpox is considered the ideal biowarfare agent [Antiviral Res. 57 (2002) 1]. Infection normally occurs after implantation of the virus on the oropharyngeal or respiratory mucosa [J. Am. Med. Assoc. 281 (1999) 2127]. Smallpox virus can be detected from the throats of exposed individuals prior to onset of illness and prior to the infectious stage of the illness. A rapid, sensitive real-time assay to detect Variola virus (smallpox) has been developed using the Vaccinia virus, a surrogate of smallpox, as a target. Cyanine 5 dye-labeled anti-Vaccinia antibody was used in a sandwich immunoassay to produce a fluorescent signal in the presence of the Vaccinia virus. The signal was detected using the Analyte 2000 biosensor (Research International, Monroe, WA). The Analyte 2000 uses a 635 nm laser diode to provide excitation light that is launched into a polystyrene optical waveguide. Fluorescent molecules within the evanescent wave are excited and a portion of their emission energy recouples into the waveguide. A photodiode quantifies the emission light at wavelengths between 670 and 710 nm. The biosensor was able to detect a minimum of 2.5 x 10(5) pfu/ml of Vaccinia virus in seeded throat culture swab specimens.

Rapid detection of Bacillus anthracis spores directly from powders with an evanescent wave fiber-optic biosensor.

There currently are no rapid, sensitive tests to directly and reliably detect Bacillus anthracis spores in common powders. Traditional culture is time consuming and molecular techniques cannot directly process powders. This study describes a biosensor assay that detects B. anthracis at concentrations of 3.2 x 10(5) spores/mg or higher in spiked powders in less than 1 h with minimal sample preparation.

Confirmation of viable E. coli O157:H7 by enrichment and PCR after rapid biosensor detection.

Many rapid tests have been developed for the detection of Escherichia coli O157:H7 from complex matrices such as food and water. However, many of these methods rely on traditional culture steps for confirmation, which can take an extra 24-48 h. The fiber optic biosensor has been used to rapidly detect pathogens from complex matrices. In this paper, we demonstrate a method using a rapid biosensor assay, recovery through a short enrichment, and PCR to detect and confirm the presence of at least 10(3) CFU/ml of E. coli O157:H7 in a sample in less than 10 h.

A rapid and automated fiber optic-based biosensor assay for the detection of Salmonella in spent irrigation water used in the sprouting of sprout seeds.

Recent outbreaks of foodborne illness have been linked to the consumption of contaminated sprouts. The spent irrigation water used to irrigate sprouts can carry many microorganisms, including pathogenic strains of Escherichia coli and Salmonella enterica. These pathogens are believed to originate from the seeds. The U.S. Food and Drug Administration recommends that sprout producers conduct microbiological testing of spent irrigation water from each production lot at least 48 h after seeds have germinated. Microbial analysis for the detection of Salmonella is labor-intensive and takes days to complete. A rapid and automated fiber-optic biosensor assay for the detection of Salmonella in sprout rinse water was developed in this study. Alfalfa seeds contaminated with various concentrations of Salmonella Typhimurium were sprouted. The spent irrigation water was assayed 67 h after alfalfa seed germination with the RAPTOR (Research International, Monroe, Wash.), an automated fiber optic-based detector. Salmonella Typhimurium could be positively identified in spent irrigation water when seeds were contaminated with 50 CFU/g. Viable Salmonella Typhimurium cells were also recovered from the waveguides after the assay. This biosensor assay system has the potential to be directly connected to water lines within the sprout-processing facility and to operate automatically, requiring manual labor only for preventative maintenance. Therefore, the presence of Salmonella Typhimurium in spent irrigation water could be continuously and rapidly detected 3 to 5 days before the completion of the sprouting process.

Detection of Escherichia coli O157:H7 in 10- and 25-gram ground beef samples with an evanescent-wave biosensor with silica and polystyrene waveguides.

A portable evanescent-wave fiber-optic biosensor was used to detect Escherichia coli O157:H7 in seeded 10- and 25-g ground beef samples. The biosensor works by launching light from a 635-nm laser diode into specially designed optical fiber probes, generating an evanescent field that extends approximately 1,000 nm from the fiber surface. Fluorescent molecules within the evanescent field are excited, and a portion of their emission recouples into the fiber probe. The return path emission is transported by an optical fiber to a photodiode within the biosensor that detects and quantifies the fluorescent signal. A sandwich immunoassay was performed on the fiber probes with cyanine 5 dye-labeled polyclonal anti-E. coli O157:H7 antibodies for generation of the specific fluorescent signal. Biotin-streptavidin interactions were used to attach polyclonal anti-E. coli O157:H7 antibodies to the surface of the fiber probe. A centrifugation method was developed to obtain samples suitable for biosensor analysis from 10- and 25-g ground beef samples. The assay was shown to be sensitive and repeatable. One hundred percent correct identification of positive samples was demonstrated at 9.0 x 10(3) CFU/g for 25-g ground beef samples with silica waveguides and at 5.2 x 10(2) CFU/g for 10-g ground beef samples with polystyrene waveguides. The reaction was highly specific. No false positives were observed for 10-g ground beef samples not spiked with the pathogen. In addition, when samples were spiked with high concentrations of a variety of non-E. coli O157:H7 organisms, no false positives were observed. The method was rapid, with results being obtained within 25 min of sample processing.

Optimization of a fluorescence sandwich enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 in apple juice.

Sandwich enzyme-linked immunosorbent assay, especially when coupled with biosensor technology, is a simple methodology that can rapidly screen juices for Escherichia coli O157:H7 contamination. However, sampling directly from apple juice and ciders has been postulated to reduce immunoassay sensitivity. In fluorescence sandwich enzyme-linked immunosorbent assays using commercially available polyclonal or monoclonal antibodies, sampling pasteurized apple juice spiked with E. coli O157:H7 compared to spiked phosphate-buffered saline shifted the range of detection. The spiked apple juice range of detection was 10(4) to 10(6) CFU/ml, whereas that for spiked phosphate-buffered saline was 10(6) to 10(8) CFU/ml, representing a hundredfold difference in sensitivity. Apple juice also increased background fluorescence intensity (P < 0.001) while reducing the net fluorescence intensity per CFU (P < 0.001). The addition of the polymer polyvinylpyrrolidone to apple juice significantly improved assay performance by increasing sensitivity and net fluorescence intensity per CFU and by reducing background fluorescence. Adjusting pH of apple juice from 3.9 to 7.4 improved assay performance but not to the degree seen with phosphate-buffered saline or polyvinylpyrrolidone-treated apple juice samples. The apple juice polyphenol, epicatechin, reduced net fluorescence intensity in a concentration-dependent manner, a change that was reversed by polyvinylpyrrolidone. Taken all together, these results suggest that polyvinylpyrrolidone can improve detection of O157:H7 in juices by reducing the effect of polyphenols on fluorescence sandwich enzyme-linked immunosorbent assay performance.

Laboratory and pilot-scale dead-end ultrafiltration concentration of sanitizer-free and chlorinated lettuce wash water for improved detection of Escherichia coli O157:H7.

An automated dead-end (single pass, no recirculation) ultrafiltration device, the Portable Multi-use Automated Concentration System (PMACS), was evaluated as a means to concentrate Escherichia coli O157:H7 from 40 liters of simulated commercial lettuce wash water. The assessment included generating, sieving, and concentrating sanitizer-free lettuce wash water, either uninoculated or inoculated with green fluorescent protein-transformed E. coli O157:H7 at a high (1.00 log CFU/ml) or low (-1.00 log CFU/ml) concentration. Cells collected within the filters were recovered in approximately 400 ml of buffer to create lettuce wash retentates. The extent of concentration was determined by viable plate counts using a medium selective for the transformed E. coli O157:H7. The samples were qualitatively analyzed for E. coli O157:H7 according to the U. S. Food and Drug Administration Bacteriological Analytical Manual enrichment method and with an electrochemiluminescence immunoassay. This concentration method was then evaluated in a pilot-scale production line at Michigan State University using chlorinated (100, 30, and 10 ppm of available chlorine) lettuce wash water. The total PMACS processing times were 82 ± 6 and 65 ± 5 min for sanitizer-free and chlorinated washes, respectively. Overall, E. coli O157:H7 populations were approximately 2 log higher in retentates than in unconcentrated lettuce wash samples. The higher E. coli O157:H7 levels in the retentates enabled cultural and electrochemiluminescence immunoassay detection in some samples when the corresponding lettuce wash samples were negative. When combined with standard and rapid detection methods, the PMACS concentration method may provide a means to enhance pathogen monitoring of produce wash water.