Poly(indole-5-carboxylic acid)/reduced graphene oxide/gold nanoparticles/phage-based electrochemical biosensor for highly specific detection of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis is an enteric bacterium causing yersiniosis in humans. The existing Yersinia pseudotuberculosis detection methods are time-consuming, requiring a sample pretreatment step, and are unable to discriminate live/dead cells. The current work reports a phage-based electrochemical biosensor for rapid and specific detection of Yersinia pseudotuberculosis. The conductive poly(indole-5-carboxylic acid), reduced graphene oxide, and gold nanoparticles are applied for surface modification of the electrode. They possess ultra-high redox stability and retain 97.7% of current response after performing 50 consecutive cycles of cyclic voltammetry.The specific bacteriophages vB_YepM_ZN18 we isolated from hospital sewage water were immobilized on modified electrodes by Au-NH2 bond between gold nanoparticles and phages. The biosensor fabricated with nanomaterials and phages were utilized to detect Yersinia pseudotuberculosis successfully with detection range of 5.30 × 102 to 1.05 × 107 CFU mL-1, detection limit of 3 CFU mL-1, and assay time of 35 min. Moreover, the biosensor can specifically detect live Yersinia pseudotuberculosis without responding to phage-non-host bacteria and dead Yersinia pseudotuberculosis cells. These results suggest that the proposed biosensor is a promising tool for the rapid and selective detection of Yersinia pseudotuberculosis in food, water, and clinical samples.

Rapid and accurate detection of Escherichia coli O157:H7 in beef using microfluidic wax-printed paper-based ELISA.

Escherichia coli O157:H7 is a severe foodborne pathogen that causes lots of life-threatening diseases. In the search for a rapid, sensitive, portable and low-cost method to detect this pathogen, we developed a wax-printed paper-based enzyme-linked immunosorbent assay (P-ELISA) based on microfluidic paper-based analytical devices (µPADs), with the whole operation time being less than 3 h and only needing 5 µl samples for detection. The limit of detection (LOD) of E. coli O157:H7 reached 104 CFU ml-1, which is an order of magnitude higher than that of conventional ELISA (C-ELISA). The LOD in artificially contaminated beef samples is 1 CFU per 25 g after enriching the culture for 8 h. This method is superior to the molecular biology method in detection sensitivity and superior to C-ELISA and the national standard method in detection time and cost. Thus, the established P-ELISA method has good sensitivity, specificity and repeatability. It can be suitable for point-of-care testing without expensive and bulky instruments and can also provide a platform for detecting other pathogens, especially in areas that lack advanced clinical equipment.

A portable microfluidic platform for rapid determination of microbial load and somatic cell count in milk.

Microfluidics systems that have been emerged in the last 20 years and used for processing the fluid in a microchannel structure at microliter levels are alternative to the conventional methods. The objective of the study is to develop a microfluidic platform for determination of the microbial load and the number of somatic cells in milk. For this purpose, a polydimethylsiloxane (PDMS) chip with a channel size of 300 µm × 60 µm was produced. Cells/bacteria labeled with fluorescent stain in milk were counted with the proposed microfluidic platform and the results were compared with the reference cell concentration/the bacterial counts by conventional method. It was found that our platform could count somatic and bacterial cells with an accuracy above 80% in 20 min run for each analysis. The portable overall platform has an overall dimension of 25x25x25 cm and weighs approximately 9 kg.

Cost-Effective Live Cell Density Determination of Liquid Cultured Microorganisms.

Live monitoring of microorganisms growth in liquid medium is a desired parameter for many research fields. A wildly used approach for determining microbial liquid growth quantification is based on light scattering as the result of the physical interaction of light with microbial cells. These measurements are generally achieved using costly table-top instruments; however, a live, reliable, and straight forward instrument constructed using parts that are inexpensive may provide opportunities for many researchers. Here, such an instrument has been constructed and tested. It consists of modular test tube holding chambers, each with a low power monochromatic light-emitting diode, and a monolithic photodiode. A microcontroller connects to all modular chambers to control the diodes, and send the live data to either an LCD screen, or a computer. This work demonstrate that this modular instrument can determine precise cell concentrations for the bacteria Escherichia coli and Pseudomonas syringae pv. tomato DC3000, as well as Saccharomyces cerevisiae yeast.

Improved detection of clinically relevant wound bacteria using autofluorescence image-guided sampling in diabetic foot ulcers.

Clinical wound assessment involves microbiological swabbing of wounds to identify and quantify bacterial species, and to determine microbial susceptibility to antibiotics. The Levine swabbing technique may be suboptimal because it samples only the wound bed, missing other diagnostically relevant areas of the wound, which may contain clinically significant bacteria. Thus, there is a clinical need to improve the reliability of microbiological wound sampling. To address this, a handheld portable autofluorescence (AF) imaging device that detects bacteria in real time, without contrast agents, was developed. Here, we report the results of a clinical study evaluating the use of real-time AF imaging to visualise bacteria in and around the wound bed and to guide swabbing during the clinical assessment of diabetic foot ulcers, compared with the Levine technique. We investigated 33 diabetic foot ulcers (n = 31 patients) and found that AF imaging more accurately identified the presence of moderate and/or heavy bacterial load compared with the Levine technique (accuracy 78% versus 52%, P = 0·048; adjusted diagnostic odds ratio 7·67, P < 0·00022 versus 3·07, P = 0·066) and maximised the effectiveness of bacterial load sampling, with no significant impact on clinical workflow. AF imaging may help clinicians better identify the wound areas with clinically significant bacteria, and maximise sampling of treatment-relevant pathogens.

Comparative Evaluation of Inoculation of Urine Samples with the Copan WASP and BD Kiestra InoqulA Instruments.

This study evaluated the quantitative results from and quality of the inoculation patterns of urine specimens produced by two automated instruments, the Copan WASP and the BD InoqulA. Five hundred twenty-six urine samples submitted in 10-ml canisters containing boric acid were processed within 30 min on an InoqulA instrument plating 10 µl of specimen, and on two WASP instruments, one plating 1 µl of specimen (WASP-1), and the second plating 10 µl of WASP (WASP-10). All samples were incubated, analyzed, and digitally imaged using the BD Kiestra total lab automation system. The results were evaluated using a quantitative protocol and assessed for the presence or absence of ≥5 distinct colonies. Separate studies were conducted using quality control (QC) organisms to determine the relative accuracy of WASP-1, WASP-10, and InoqulA instruments compared to the results obtained with a calibrated pipette. The results with QC organisms were calculated as the ratios of the counts of the automated instruments divided by the counts for the calibrated pipette (the gold standard method). The ratios for the InoqulA instrument were closest to 1.0, with the smallest standard deviations indicating that compared to a calibrated pipette, the InoqulA results were more accurate than those with the WASP instrument. For clinical samples, the WASP instruments produced higher colony counts and more commensals than the InoqulA instrument, with differences most notable for WASP-1. The InoqulA instrument was significantly better at dispersing organisms with counts of ≥10(5) bacteria/ml of urine than were the WASP-1 and WASP-10 instruments (P < 0.05). Our results suggest that the InoqulA quantitative results are more accurate than the WASP results, and, moreover, the number of isolated colonies produced by the InoqulA instrument was significantly greater than that produced by the WASP instrument.

A disposable bacterial lysis cartridge (BLC) suitable for an in situ water-borne pathogen detection system.

We constructed a disposable bacterial lysis cartridge (BLC) suitable for an in situ pathogen detection system. It had an in-built micro corona discharge based ozone generator that provided ozone for cell lysis. Using a custom sample handling platform, its performance was evaluated with a Gram-positive bacterium of Bacillus subtilis. It was capable of achieving a similar degree of lysis as a commercial ultrasonic dismembrator with a P-1 microprobe in 10 min at an air pump flow rate of 29.4 ml min(-1) and an ozone generator operating voltage of 1600 V. The lysing duration could be significantly reduced to 5 min by increasing the air pump flow rate and the ozone generator operating voltage as well as by the addition of sodium dodecyl sulfate (SDS).

A new device for the prompt diagnosis of urinary tract infections.

BACKGROUND: Urinary tract infections (UTIs) are among the most common infectious diseases. RESULTS obtained from conventional microbiological analysis of urine and antibiotic susceptibility testing are available only after a few days, delaying precise diagnosis and appropriate therapy. Micro Biological Survey (MBS) srl (a spin-off of Roma Tre University, Rome, Italy) has developed and patented an automated colorimetric test for rapid bacterial counting. In a preliminary validation study it was demonstrated that the results obtained with the MBS method are equivalent to the results obtained with conventional culture-based microbiological analysis. METHODS: In this study, sterile urine samples were artificially contaminated with bacterial species that are most frequently responsible for UTIs. The MBS method was used to evaluate the presence of bacteria and their sensitivity to some of the most commonly used antibiotics in UTIs. RESULTS: The MBS method was able to detect in a few hours the presence or absence of bacteria at clinically significant concentrations (>105 CFU/mL), and to provide their susceptibility pattern to a limited panel of antibiotics. DISCUSSION: The results obtained demonstrate that the MBS point-of-care testing (POCT) device could be developed into a valuable aid for the management of UTIs, possibly addressing more precise diagnosis and appropriate therapy.

Mobile phones in clinical practice: reducing the risk of bacterial contamination.

BACKGROUND: Mobile smart phones have become increasingly integrated into the practice of doctors and allied medical professionals. Recent studies suggest them to represent reservoirs for pathogens with potential to cause nosocomial infections. This study aimed to investigate the level of contamination on phones used on surgical wards and identify strategies for their safe use within clinical areas. METHODS: Fifty mobile phones were taken from members of the multidisciplinary team working in a surgical unit. Phones were swabbed by two trained investigators using a standardised technique and samples streaked out using an automated specimen inoculator onto two types of culture media (Columbia blood agar and MacConkey agar). Colonies were identified and counted by a single trained investigator in a blinded fashion. Simultaneously a questionnaire investigating usage levels of phones was given to 150 healthcare workers. RESULTS: Sixty per cent of phones sampled had some form of contaminant isolated from their phone. Thirty-one (62%) of phones had only three colonies or less isolated on medium. No pathogenic or drug resistant strains of bacteria were identified. A total of 88% of individuals sampled by questionnaire used their phone within the workplace of which 55% used it for clinical purposes. Sixty-three per cent expected there to be some form of contaminant on their phone with only 37% admitting to cleaning it regularly. Seventy-five per cent of people did not view a ban on phones as a practical solution was they found to be an infection risk. CONCLUSION: Touch screen smart phones may be used safely in a clinical environment, with a low risk of cross-contamination of nosocomial bacteria to patients, in the setting of effective adherence to hand hygiene policies.

Bacteria species identification by the statistical analysis of bacterial colonies Fresnel patterns.

It was demonstrated that statistical analysis of bacteria colonies Fresnel patterns recorded in the optical system with converging spherical wave illumination is suitable for highly effective bacteria species classification. The proposed method includes Fresnel patterns recording followed by image processing and the statistical analysis based on feature extraction, feature selection, classification and classification performance methods. Examination performed on various bacteria species (Salmonella enteritidis, Staphylococcus aureus, Staphylococcus intermedius, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa and Citrobacter freundii) revealed that the proposed method achieved very high accuracy of over 98%.

Selective enumeration of propionibacteria in Emmental-type cheese using Petrifilm™ aerobic count plates added to lithium glycerol broth.

Propionibacteria derived from dairy products are relevant starter cultures for the production of Swiss and Emmental-type cheeses, and the monitoring of which is mandatory for proper quality control. This study aimed to evaluate an alternative procedure to enumerate propionibacteria, in order to develop a reliable and practical methodology to be employed by dairy industries. 2,3,5-triphenyltetrazolium chloride (TTC) inhibitory activity was tested against five reference strains (CIRM 09, 38, 39, 40 and 116); TTC at 0·0025% (w/v) was not inhibitory, with the exception of one strain (CIRM 116). Subsequently, the four TTC-resistant strains, three commercial starter cultures (PS-1, PB-I, and CHOO) and twelve Emmental-type cheese samples were subjected to propionibacteria enumeration using Lithium Glycerol (LG) agar, and Petrifilm™ Aerobic Count (AC) plates added to LG broth (anaerobic incubation at 30 °C for 7 d). Petrifilm™ AC added to LG broth presented high counts than LG agar (P<0·05) for only two reference strains (CIRM 39, and 40) and for all commercial starter cultures. Cheese sample counts obtained by both procedures did not show significant differences (P<0·05). Significant correlation indexes were observed between the counts recorded by both methods (P<0·05). These results demonstrate the reliability of Petrifilm™ AC plates added to LG broth in enumerating select Propionibacterium spp., despite some limitations observed for specific commercial starter cultures.

A new objective evaluation method for PU cleansing using a rapid bacteria counting system.

OBJECTIVE: To assess a new rapid bacterial counting device for evaluating wound cleansing effectiveness based on dielectrophoretic impedance measurement. METHOD: Three patients with pressure ulcers with undermining were recruited, and pressure ulcer severity assessed using the DESIGN-R tool. The number of bacteria was measured using this new apparatus both before and after wound cleansing, performed by wound, ostomy, and continence nurses using apH-balanced cleanser for periwound skin and with normal saline for the wound bed and undermining area. RESULTS: The results showed that wound cleansing effectively reduced bacterial counts on the periwound skin, wound bed and undermined site, with a median number of bacteria of 3.6×106 CFU/ml before cleansing, which decreased to 1.1×106 CFU/ml after cleansing. CONCLUSION: This pilot study result suggests the usefulness of this new device for assessing the effectiveness of wound cleansing on reduction of bacterial number. Standardisation of wound cleansing technique may be achieved by hands-on education using this apparatus at bedside.

Rapid detection of viable microorganisms based on a plate count technique using arrayed microelectrodes.

Development of a miniaturized biosensor system that can be used for rapid detection and counting of microorganisms in food or water samples is described. The developed microsystem employs a highly sensitive impedimetric array of biosensors to monitor the growth of bacterial colonies that are dispersed across an agar growth medium. To use the system, a sample containing the bacteria is cultured above the agar layer. Using a multiplexing network, the electrical properties of the medium at different locations are continuously measured, recorded, and compared against a baseline signal. Variations of signals from different biosensors are used to reveal the presence of bacteria in the sample, as well as the locations of bacterial colonies across the biochip. This technique forms the basis for a label-free bacterial detection for rapid analysis of food samples, reducing the detection time by at least a factor of four compared to the current required incubation times of 24 to 72 hours for plate count techniques. The developed microsystem has the potential for miniaturization to a stage where it could be deployed for rapid analysis of food samples at commercial scale at laboratories, food processing facilities, and retailers.

Discrimination of Escherichia coli strains using glycan cantilever array sensors.

Carbohydrate-based sensors, that specifically detect sugar binding molecules or cells, are increasingly important in medical diagnostic and drug screening. Here we demonstrate that cantilever arrays functionalized with different mannosides allow the real-time detection of several Escherichia coli strains in solution. Cantilever deflection is thereby dependent on the bacterial strain studied and the glycan used as the sensing molecule. The cantilevers exhibit specific and reproducible deflection with a sensitivity range over four orders of magnitude.

Evaluation of in-office dental unit waterline testing.

In-office dental unit waterline (DUWL) testing systems are commercially available for monitoring DUWL bacteria. The current study compared Aquasafe, Petrifilm, and Heterotrophic Plate Count Sampler (HPCS) with R2A plating methodology, considered the gold standard for enumerating heterotrophic bacteria in potable water. Samples were collected from 20 dental units. Heterotrophic bacterial counts of ≤500 CFUs/mL were used as the cut-off for assessing in-office testing compared to R2A laboratory plating. Validity was assessed using sensitivity and specificity, along with positive and negative predictive values. Results were also compared using concordance and kappa statistics. All in-office tests demonstrated 100% specificity and positive predictive values, while sensitivity and negative predictive values were low (Petrifilm, 57%/50%; HPCS, 50%/46%; Aquasafe, 21%/35%). Concordance and kappa values for agreement with R2A plating were as follows: Petrifilm 70% (κ = 0.44), HPCS 65% (κ = 0.38), and Aquasafe 45% (κ = 0.14). In-office DUWL testing with Aquasafe, Petrifilm, and HPCS agreed poorly with R2A plating methodology and is not valid or reliable as a means of accurately monitoring bacterial density in DUWL. These in-office test systems should not be used for assessing compliance with the ADA and CDC standard for acceptable heterotrophic bacterial counts in DUWLs (≤500 CFUs/mL).

Colorimetric bacteria sensing using a supramolecular enzyme-nanoparticle biosensor.

Rapid and sensitive detection of pathogens is a key requirement for both environmental and clinical settings. We report here a colorimetric enzyme-nanoparticle conjugate system for detection of microbial contamination. In this approach, cationic gold nanoparticles (NPs) featuring quaternary amine headgroups are electrostatically bound to an enzyme [ß-galactosidase (ß-Gal)], inhibiting enzyme activity. Analyte bacteria bind to the NP, which releases the ß-Gal and restores its activity, providing an enzyme-amplified colorimetric readout of the binding event. Using this strategy, we have been able to quantify bacteria at concentrations of 1 × 10(2) bacteria/mL in solution and 1 × 10(4) bacteria/mL in a field-friendly test strip format.

Rapid, semiautomated quantification of bacterial cells in freshwater by using a microfluidic device for on-chip staining and counting.

A microfluidic device-based system for the rapid and semiautomated counting of bacteria in freshwater was fabricated and examined. Bacteria in groundwater and in potable water, as well as starved Escherichia coli O157:H7 spiked in pond water, were able to be on-chip stained and enumerated within 1 h using this system.

Detection of bacteria using bacteriophages as recognition elements immobilized on long-period fiber gratings.

The paper presents for the first time a study of long-period gratings (LPGs) applied for label-free detection of specific bacteria using physically adsorbed bacteriophages. For the purposes of the experiment a number of highly sensitive LPGs working at the turning point of phase matching curve was fabricated in SMF28 fiber using UV exposure. We show that the device allows for real-time monitoring of phenomena taking place on the sensor's surface, including phage-bacteria interactions. For the applied conditions a resonance wavelength shift of ~1.3 nm induced by bacteria binding was observed.

Assessment of the application of an automated electronic milk analyzer for the enumeration of total bacteria in raw goat milk.

Automated electronic milk analyzers for rapid enumeration of total bacteria counts (TBC) are widely used for raw milk testing by many analytical laboratories worldwide. In Ontario, Canada, Bactoscan flow cytometry (BsnFC; Foss Electric, Hillerød, Denmark) is the official anchor method for TBC in raw cow milk. Penalties are levied at the BsnFC equivalent level of 50,000 cfu/mL, the standard plate count (SPC) regulatory limit. This study was conducted to assess the BsnFC for TBC in raw goat milk, to determine the mathematical relationship between the SPC and BsnFC methods, and to identify probable reasons for the difference in the SPC:BsnFC equivalents for goat and cow milks. Test procedures were conducted according to International Dairy Federation Bulletin guidelines. Approximately 115 farm bulk tank milk samples per month were tested for inhibitor residues, SPC, BsnFC, psychrotrophic bacteria count, composition (fat, protein, lactose, lactose and other solids, and freezing point), and somatic cell count from March 2009 to February 2010. Data analysis of the results for the samples tested indicated that the BsnFC method would be a good alternative to the SPC method, providing accurate and more precise results with a faster turnaround time. Although a linear regression model showed good correlation and prediction, tests for linearity indicated that the relationship was linear only beyond log 4.1 SPC. The logistic growth curve best modeled the relationship between the SPC and BsnFC for the entire sample population. The BsnFC equivalent to the SPC 50,000 cfu/mL regulatory limit was estimated to be 321,000 individual bacteria count (ibc)/mL. This estimate differs considerably from the BsnFC equivalent for cow milk (121,000 ibc/mL). Because of the low frequency of bulk tank milk pickups at goat farms, 78.5% of the samples had their oldest milking in the tank to be 6.5 to 9.0 d old when tested, compared with the cow milk samples, which had their oldest milking at 4 d old when tested. This may be one of the major factors contributing to the larger goat milk BsnFC equivalence. Correlations and interactions between various test results were also discussed to further understand differences between the 2 methods for goat and cow milks.

A microfluidic device for bacteria detection in aqueous samples.

We demonstrate the use of a microfluidic device that accurately monitors bacteria levels in water. Fluorescent antibody labelled E. coli K12 bacteria in aqueous samples are flowed through the device, and spikes in the emission signal correspond to the presence of bacteria. Target bacteria could also be detected while suspended in a suspension of very low antibody concentration, suggesting that washing of bacteria could be avoided to make the detection automated and more rapid. Use of a microfluidic device will eventually enable quick on-site detection of bacteria using small quantities of sample.