Dead-end ultrafiltration concentration and IMS/ATP-bioluminescence detection of Escherichia coli O157:H7 in recreational water and produce wash

The purpose of this study was to develop a detection method for viable E. coli O157:H7 in fresh produce and recreational water. The method was evaluated using eight samples of produce wash and recreational water with or without spiked E. coli O157:H7 at ≤ 10² CFU·ml^− 1 and concentrated using dead-end ultrafiltration (DEUF) to produce primary and secondary retentates. Fifty-four matrix replicates of undiluted secondary retentates or dilutions (1:2 or 1:10 in buffer) were evaluated using an IMS/ATP bioluminescence assay (IMS/ATP). Combining primary and secondary DEUF yielded a 2-4 log₁₀ increase in E. coli O157:H7 concentrations in spiked samples and resulted in signal-to-noise ratios 2-219 fold higher than controls, depending on the sample type. DEUF increased the concentration of E. coli O157:H7 to within the detectable limits of IMS/ATP. The combined assay provided detection of viable E. coli O157:H7 in produce and recreational water. Accurate detection of microbial pathogens using DEUF and IMS/ATP could reduce disease outbreaks from contaminated water sources and food products.     

Application of ion mobility spectrometry to the rapid screening of methamphetamine incorporated in hair

Using ion mobility spectrometry (IMS), a simple, sensitive and rapid screening for methamphetamine (MA) incorporated in user's hair has been developed. To completely unbind MA from hair matrix and to achieve its effective vaporization for the IMS detection, the hair sample was digested in 5 M NaOH (methanol-water, 4:1, v/v) solution prior to IMS measurement. MA in hair was semi-quantitatively detected by monitoring the digested hair sample employing dibenzylamine (DBA) as internal standard. The minimum amount of hair sample required was 2 mg and its digested sample was ample for four IMS measurements. Teh detection limit of MA in hair was 0.5 ng mg⁻¹. This proposed method was applicable to the semi-quantitative detection of MA in users' hair samples, and to the sectional analysis for MA in a limited amount of user's hair. The IMS results obtained were in good agreement with their GC-MS determination.     

Evaluation of cyst loss in standard procedural steps for detecting ofGiardia lamblia andCryptosporidium parvum in water

The standard procedure outlined by the United States Environmental Protection Agency (US EPA) in Method 1623 for analyzingGiardia lamblia cysts andCryptosporidium parvum oocysts in water samples consists of filtration, elution, centrifugal concentration, immunomagnetic separation (IMS), and immunofluorescence assay (IFA) followed by microscopic examination. In this study, the extent of (oo)cyst loss in each step of this procedure was evaluated by comparing recovery yields in segmented analyses: (i) IMS+IFA, (ii) concentration +IMS+IFA, and (iii) filtration/elution + concentration +IMS+IFA. The complete (oo)cyst recovery by the full procedure was 52–57%. The (oo)cyst loss in the IMS step was only 0–6%, implying that IMS is a fairly reliable method for (oo)cyst purification. Centrifugal concentration of the eluted sample and pellet collection before IMS resulted in a loss of 8–14% of the (oo)cysts. The largest (oo)cyst loss occurred in the elution step, with 68–71% of the total loss. The permeated loss of (oo)cysts was negligible during filtration of the water sample with a 1.0-μm pore polyethersulfone (PES) capsule. These results demonstrated that the largest fraction of (oo)cyst loss in this procedure occurred due to poor elution from the filter matrix. Improvements in the elution methodology are therefore required to enhance the overall recovery yield and the reliability of the detection of these parasitic protozoa.     

Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r2 = 0. 95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.     

Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef

In this study, enrichment procedures and two recovery methods, a membrane surface adhesion technique and an immunomagnetic separation (IMS), were compared for use in conjunction with a multiplex polymerase chain reaction (PCR) method with a view to describing a fast (24 h) and economical test for detection of Escherichia coli O157:H7 in meat samples. The study showed no significant difference between three different enrichment media (BHI, E. coli (E.C.) broth+novobiocin, modified tryptone soya broth (mTSB)+novobiocin) or two incubation temperatures (37 or 41.5 degrees C) for growth of E. coli O157:H7 in minced beef. Minced beef samples inoculated with E. coli O157:H7 at 40 cfu g(-1) were incubated at 37 degrees C for 16 h in E.C. broth+novobiocin reaching numbers of (log(10)7.82-8.70). E. coli O157:H7 were recovered by attachment to polycarbonate membranes immersed in the enriched cultures for 15 min or by immunomagnetic separation. Subsequent treatment of recovered membranes or IMS beads with lysis buffer and phenol/chloroform/isoamyl alcohol was used to extract the DNA from the extracted E. coli O157:H7 cells. The results show when E. coli O157:H7 was present at high levels in the enriched meat sample (log(10)9.6-7.5 cfu ml(-1); >16-h enrichment), the membrane and IMS techniques recovered similar levels of the pathogen and the microorganism was detectable by PCR using both methods. At lower levels of E. coli O157:H7 (log(10)6.4), only the IMS method could recover the pathogen but at levels below this neither method could recover sufficient numbers of the pathogens to allow detection. The conclusion of the study is that with sufficient enrichment time (16 h) the membrane surface adhesion membrane extraction method used in combination with multiplex PCR has the potential for a rapid and economical detection method.     

Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef

AIM: To develop an improved, rapid and sensitive sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef. METHODS AND RESULTS: Fresh ground beef samples were experimentally inoculated with varying concentrations of E. coli O157:H7. PCR inhibitors were removed and bacterial cells were concentrated by filtration and centrifugation, and lysed using enzymatic digestion and successive freeze/thaw cycles. DNA was purified and concentrated via phenol/chloroform extraction and the Shiga toxin 1 gene (stx1) was amplified using PCR to evaluate the sample preparation method. Without prior enrichment of cells in broth media, the detection limit was 103 CFU g-1 beef. When a 6 h enrichment step was incorporated, the detection limit was 1 CFU g-1 beef. The total time required from beginning to end of the procedure was 12 h. CONCLUSIONS: The sample preparation method developed here enabled substantially improved sensitivity in the PCR-based detection of E. coli O157:H7 in ground beef, as compared to previous reports. SIGNIFICANCE AND IMPACT OF THE STUDY: Superb sensitivity, coupled with quick turn-around time, relative ease of use and cost-effectiveness, makes this a useful method for detecting E. coli O157:H7 in ground beef.     

Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

Aims:  The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water.
Methods and Results:  Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis ( r  = 0·62 and 0·77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci.
Conclusions:  The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria.
Significance and Impact of the Study:  The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications.     

Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623

The U.S. Environmental Protection Agency has developed method 1623 for simultaneous detection of Cryptosporidium oocysts and Giardia cysts in water. Method 1623 includes four major steps: filtration, immunomagnetic separation (IMS), fluorescent antibody (FA) staining and microscopic examination. It was noted that the recovery levels following IMS-FA and FA staining were high, averaging more than 92.0% and 89.0% for C. parvum oocysts and G. lamblia cysts, respectively. In contrast, when the filtration step was incorporated, the recovery level of C. parvum oocysts declined significantly to 18.1% in seeded tap water, while a relatively high recovery level of 77.2% for G. lamblia cysts could still be achieved. Further study indicated that the recovery level of C. parvum oocysts could be enhanced significantly when an appropriate amount of silica particles was added to a water sample. The recovery level of C. parvum oocysts was affected by particle size and concentration. The optimal silica particle size was determined to be within the range of 5-40 microm, and the corresponding optimal silica concentration was 1.42 g for 10-l tap water. When both G. lamblia cysts and C. parvum oocysts were spiked into the tap water sample containing the optimum amount of silica particles, the average recovery levels of oocysts and cysts were 82.7% and 75.4%, respectively. The results obtained clearly suggested that addition of an appropriate amount of silica particles could improve the recovery level of C. parvum oocysts significantly and yet there was no noticeable deleterious effect on the recovery level of G. lamblia cysts. Further study indicated that the rotation time in the IMS procedure using the Dynal GC-Combo IMS kit (which was recommended in method 1623) was important for G. lamblia cyst detection. In contrast, the recovery level of C. parvum oocysts was not affected by the rotation time. Furthermore, it was found that the recovery levels of C. parvum oocysts using methods 1622 and 1623 were quite close although different IMS kits were used in the two methods.     

Immunomagnetic-electrochemiluminescent detection of Escherichia coli O157 and Salmonella typhimurium in foods and environmental water samples.

Hemorrhagic Escherichia coli O157:H7 strains and other virulent enteric pathogens can pose a serious health threat in tainted meats, poultry, and even drinking water. Traditional culture-based methods for assay of enteric pathogens in foods and water sources are relatively slow, and results can be ambiguous. Immunomagnetic separation (IMS) and detection methods have been investigated and appear promising for rapid bacterial assay of foods and environmental samples. In this work, a commercial sensor which combines IMS with electrochemiluminescence (ECL) detection is evaluated for detection of E. coli O157 and Salmonella typhimurium in foods and fomites. Results indicate that detection limits are in the range of 100 to 1,000 bacteria per ml in pristine buffer for E. coli O157 and S. typhimurium, respectively, or 1,000 to 2,000 bacteria per ml in food samples (depending on the sample) and that total processing and assay time is rapid (< 1 h) even in food samples. An immunologic "hook" or high-antigen-concentration prozone effect was observed above 10(4) and 10(5) bacteria per ml for E. coli O157 and S. typhimurium, respectively. IMS was accomplished in milk, juices, serum, supernatant fluids from ground beef, finely minced chicken, and fish suspensions as well as several freshwater sources and followed by ECL assay. Some samples, especially fish, gave unexpectedly high background ECL. Conversely, low ECL intensity was observed in nonfat and 2% fat milk samples, which appeared to be related to binding or entrapment of the antibody-coated magnetic beads by particulates in the milk, as revealed by microscopy. Results of this evaluation suggest the feasibility of immunomagnetic-ECL methodology for rapid, sensitive, and facile preliminary screening of various foods and fomites for the presence of virulent enteric pathogens.     

Sensitive Detection of Escherichia coli O157:H7 in Food and Water by Immunomagnetic Separation and Solid-Phase Laser Cytometry

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r² = 0.95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r² = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.     

Detection of E. coli in beach water within 1 hour using immunomagnetic separation and ATP bioluminescence

The contamination of beach waters occurs from the discharge of storm water and sanitary sewer over?ows containing faecal material. Additional faecal material derives from discharge of animals and waterfowl. In order to protect public from exposure to faecal‐contaminated water, it is required to test enteric indicators in beach water. The problem is that the traditional culture‐based methods cannot meet this goal because it takes too long (>24 h), so the results are not available until a day later. A rapid method for testing beach water for Escherichia coli within 1 h has been developed. Immunomagnetic separation (IMS) and ATP bioluminescence were used for selective capture and quanti?cation, respectively. This rapid method was compared to the current method (m‐TEC) using beach water samples. The beach samples were pre?ltered with a 20 µm pore size ?lter in order to remove algae, plant debris and large particles. The results showed that the pre?ltration step did not trap the bacteria which were present in the original water samples. The pre?ltered water was then passed through a 0.45 µm pore size ?lter for concentration. The deposited bacteria were resuspended and then mixed with superparamagnetic polystyrene beads (diameter of 0.6 µm) that were coated with E. coli antibodies. After IMS, the quanti?cation of the E. coli was done by ATP bioluminescence. The results obtained with IMS‐ATP bioluminescence correlated well with the plate count method (Rsq = 0.93). The detection limit of the assay was about 20 CFU/100 mL, which is well below the US EPA limits for recreational water. The entire procedure can be completed in less than 1 hour. The necessary equipment is portable and was tested on‐site. Copyright © 2004 John Wiley & Sons, Ltd.     

Determination of heterocyclic aromatic amines in beef extract, cooked meat and rat urine by liquid chromatography with coulometric electrode array detection

This paper describes a method for the determination of heterocyclic aromatic amines (HAs; DMIP, IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, AalphaC, PhIP) by high-performance liquid chromatography (HPLC) with coulometric electrode array detection. The compounds are separated on reversed phase columns (LiChroCart Superspher 60 RP-select B, 250 mm x 2 mm, 4 microm and LiChrospher 60 RP-select B, 250 mm x 4 mm, 5 microm) using mobile phases consisting of acetonitrile/buffer/distilled water and detected at eight working electrodes at potentials between +190 and +680 mV against modified palladium electrodes. In the context of an EU-interlaboratory exercise, the method was applied to analyse a standardised test solution and--after isolation of the analytes by several clean-up steps--for the analysis of standardised beef extract and grilled meat. Further, the method could be applied for the analysis of HAs in suspensions of bacteria and rat urine without any sample preparation step beyond sample dilution. The data obtained show that HPLC with coulometric electrode array detection gives accurate results.     

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.     

The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures

A.R. BENNETT, S. MACPHEE AND R.P. BETTS. 1996. The use of immunomagnetic separation (IMS) techniques has been reported to reduce the total test time, and improve the sensitivity, of microbiological tests done on foods. This approach is being adopted in epidemiological investigations into suspected foodborne outbreaks of Escherichia coli O157 infection and has gained acceptance by public health laboratories and the food industry. This study demonstrated the ability of a commercially available IMS procedure, Dynabeads anti- E. coli O157, to enable detection of a few cells of E. coli O157 in 25 g of inoculated minced beef, giving results 1 d earlier than a cultural analysis of similar sensitivity. With correct choice of enrichment broths, IMS may increase isolation rate of E. coli O157 compared to that obtained using conventional cultural methods. It is suggested that this may be due to an increase in relative concentration of E. coli O157 compared with the background microflora present in minced beef, which may reduce reliability of non-IMS detection procedures by masking or mimicking target cells on selective/differential solid media. The use of an immunoassay incorporating an IMS step, EHEC-Tek (Organon-Teknika), enabled detection of a few cells of E. coli O157 in 25 g of minced beef. Comparison of the IMS-ELISA with a standard ELISA procedure (Tecra) indicated the sensitivity of the latter system to be greater, perhaps resulting in the higher isolation rate. The use of a method to reliability isolate and detect extremely low levels of E. coli O157 in a food is necessary to aid reduction in the incidence of this most serious of foodborne pathogens.     

Adsorption of endogenous polyphenols relieves the inhibition by fruit juices and fresh produce of immuno-PCR detection of Escherichia coli O157:H7

The immuno-polymerase chain reaction (PCR) approaches facilitate rapid (8 h) detection of Escherichia coli O157:H7 in contaminated dairy products and ground beef samples with detection sensitivities approaching 1 colony forming unit (cfu) g-1 ml-1. However, no PCR products were obtained when the method was applied to identify E. coli O157:H7 in tainted apple juice. Enzyme-linked immuno-assay (ELISA) results suggested non-specific binding of endogenous polyphenols (ubiquitous in plant products) to antibodies present on the surface of the immunobeads, making the latter unavailable for capturing the target bacteria Treatment of the test sample, prior to IMS, with a synthetic fining agent, polyvinylpyrrolidone, restored the full function and sensitivity of the immuno-PCR. The study demonstrates the suitability of the improved method as a generic strategy for rapid screening of fruit juices and plant produce for E. coli O157:H7.     

A comparison of sensitivity between direct plate culture, immunomagnetic separation and polymerase chain reaction for the isolation of enterohemorrhagic Escherichia coli O157]

Sensitivities of direct plate culture (DPC) method, immunomagnetic separation (IMS) method, and polymerase chain reaction (PCR) assay for successful detection Escherichia coli O157 in the food samples were compared. Three lots of minced beef and three lots of radish sprout, both of which were commercially retailed, were enriched with non-selective broth media at 36 degrees C for 6 h. After enrichment, the cultures of the minced beef and those of the radish sprout were found to have background microflora at ca.10(5)-10(7) CFU/ml and ca.10(8) CFU/ml, respectively. The cultures were then experimentally inoculated with E. coli O157 strains at various final concentrations ranging from ca.10 to 10(7) CFU/ml. The samples thus prepared were subjected to the above three methods to evaluate their detection limits. For the samples of minced beef, the detection limits of the DPC method was 10(2) CFU/ml whilst that of the IMS method was ca.10 CFU/ml. For the samples of radish sprout, the detection limits of the DPC method, the IMS method, and the PCR assay were ca.10(4) CFU/ml, ca.10(2) CFU/ml, and ca.10(6) CFU/ml, respectively. There results strongly suggest that the IMS method is most sensitive method for the detection of O157 from food samples among the methods currently available.     

Development of monoclonal antibody-coated immunomagnetic beads for separation and detection of norovirus (genogroup II) in faecal extract samples

Aims:  The aim of this study is to develop an RT-PCR assay combined with immunomagnetic beads (IMS/RT-PCR) coating monoclonal antibody (Mab) for separation and detection of norovirus (genogroup II) in faecal samples. We furthermore compare its detection limits with IMS/RT-PCR using polyclonal antibody (Pab) and the TRIzol extraction method followed by RT-PCR (TRIzol-RT-PCR).
Methods and Results:  Mab-coated beads and Pab-coated beads were added to a series of tenfold dilutions of faecal extract containing norovirus in 1 ml PBS. After incubation and collection, the RNA was released by heating from virus separated by beads. The tenfold dilutions of faecal were also extracted with TRIzol reagent. The RNA was used as the template for RT-PCR detection (primers: JV12–JV13). IMS/RT-PCR using Mab showed an endpoint in the 10⁻⁷ dilution and was 10² times more sensitive than IMS/RT-PCR using Pab and was at least 10³ times more sensitive than TRIzol-RT-PCR method.
Conclusions:  IMS/RT-PCR using Mab proved to be a more sensitive method of noroviruses (NVs) detection than IMS/RT-PCR using Pab and the TRIzol-RT-PCR method.
Significance and Impact of the Study:  This is the first study to detect NVs with IMS/RT-PCR using Mab, and could serve as a model for future assays when broadly reactive NVs-specific Mabs are developed.