Immunomagnetic separation as a sensitive method for isolating<i>Escherichia coli</i>O157 from food samples

Wright, Dominic; Chapman, P. A.; Siddons, C. A.

doi:10.1017/s0950268800051438

Cited by 181 publications

(108 citation statements)

References 24 publications

(33 reference statements)

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“…Immunomagnetic separation (IMS) is another prevalent method for a relatively rapid, clean and specific separation of bacteria from liquid food samples, such as milk and juice. The IMS technique uses magnetic beads (1-5 μm) conjugated with specific antibodies which adhere to the bacterial cells and thus help to separate the bacteria in the presence of a strong magnetic field (12,60). Following aspiration of the supernatant, the bead-analyte conjugates are re-suspended in a buffer and used for detection.…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Digestion for Improved Bacteria Separation from Leafy Green Vegetables

Wang

et al. 2016

Journal of Food Protection

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An effective and rapid method for the separation of bacteria from food matrix remains a bottleneck for rapid bacteria detection for food safety. Bacteria can strongly attach to a food surface or internalize within the matrix, making their isolation extremely difficult. Traditional methods of separating bacteria from food routinely involve stomaching, blending, and shaking. However, these methods may not be efficient at removing all the bacteria from complex matrices. Here, we investigate the benefits of using enzyme digestion followed by immunomagnetic separation to isolate Salmonella from spinach and lettuce. Enzymatic digestion using pectinase and cellulase was able to break down the structure of the leafy green vegetables, resulting in the detachment and release of Salmonella from the leaves. Immunomagnetic separation of Salmonella from the liquefied sample allowed an additional separation step to achieve a more pure sample without leaf debris that may benefit additional downstream applications. We have investigated the optimal combination of pectinase and cellulase for the digestion of spinach and lettuce to improve sample detection yields. The concentrations of enzymes used to digest the leaves were confirmed to have no significant effect on the viability of the inoculated Salmonella. Results reported that the recovery of the Salmonella from the produce after enzyme digestion of the leaves was significantly higher (P <0.05) than traditional sample preparation methods to separate bacteria (stomaching and manually shaking). The results demonstrate the potential for use of enzyme digestion prior to separation can improve the efficiency of bacteria separation and increase the likelihood of detecting pathogens in the final detection assay.

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Section: Introductionmentioning

confidence: 99%

Enzymatic Digestion for Improved Bacteria Separation from Leafy Green Vegetables

Wang

et al. 2016

Journal of Food Protection

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“…Early applications of IMS were for separation of B lymphocytes from whole blood (Rasmussen et al, 1990); however, this technique has been developed further for separation of specific bacteria from faeces or food samples. For instance, IMS was more sensitive than conventional culture for detecting Escherichia coli O157 in faecal samples of cattle and sheep (Heuvelink et al, 1998) and in food samples (Wright et al, 1994). Similarly, IMS improved the separation of Salmonella Typhimurium (100 c.f.u.)…”

mentioning

confidence: 99%

Immunomagnetic separation of the intestinal spirochaetes Brachyspira pilosicoli and Brachyspira hyodysenteriae from porcine faeces

Corona-Barrera¹,

Smith²,

La³

et al. 2004

Journal of Medical Microbiology

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Porcine intestinal spirochaetes are fastidious anaerobic organisms and, as a consequence, it has been necessary to develop various protocols to enhance their isolation from or detection in faeces.Immunomagnetic separation (IMS) is a method developed recently to improve separation of target cells from mixed cell suspensions. The purpose of the present study was to compare the relative sensitivity of IMS for isolation of Brachyspira pilosicoli and Brachyspira hyodysenteriae with current routine diagnostic methods (culture on selective media and PCR) for detection of these microorganisms in pig faeces. Neither direct nor indirect IMS methods enhanced the sensitivity of detection of either organism when performed with the recommended washings during sample processing. Performance of the IMS procedure without washing gave sensitivity at levels similar to direct culture onto selective medium. Further development of IMS techniques is required to improve isolation rates of Brachyspira species from faecal samples. INTRODUCTIONIntestinal spirochaetes are mainly members of the genus Brachyspira, which includes species commensal and pathogenic for pigs and other animals and humans. Brachyspira hyodysenteriae is recognized as the aetiological agent of swine dysentery (SD) (Taylor & Alexander, 1971) and Brachyspira pilosicoli is the aetiological agent of a disease of pigs known as intestinal spirochaetosis (Taylor et al., 1980) or porcine colonic spirochaetosis (PCS;Duhamel et al., 1998). Infection with B. pilosicoli has been reported in a wide range of hosts, including dogs (Duhamel et al., 1995), chickens and other avian species (Dwars et al., 1992;McLaren et al., 1997), guinea pigs (Vanrobaeys et al., 1998), non-human primates (Takeuchi et al., 1974) and humans (Cooper et al., 1986; Surawicz et al., 1987). As interest in the field of intestinal spirochaetes has grown, further species have been identified recently as pathogens in animal species other than pigs. For instance, Brachyspira alvinipulli has been described and shown to be pathogenic for chickens (Stanton et al., 1998) and the provisionally designated 'Serpulina canis' has been found in dogs (Duhamel et al., 1998).Porcine intestinal spirochaetes are Gram-negative, anaerobic and fastidious organisms. As a result, various culture media and incubation conditions have been assessed in order to develop the most reliable and efficient method for their isolation from faecal samples. Typically, isolation of these bacteria is achieved by culture on selective media based on trypticase soy agar or Columbia agar base supplemented with blood and multiple antibiotics to reduce the growth of other bacteria. Various antibiotic combinations have been utilized including spectinomycin (TSA-S400 medium; Songer et al., 1976), spiramycin-colistin-vancomycin (TSA-CVS medium;Jenkinson & Wingar, 1981) and spectinomycin-colistinvancomycin-spiramycin-rifampicin (BJ medium; Kunkle & Kinyon, 1988). In a comparative study of these media, BJ proved to be the most efficient in eliminating norm...

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“…An initial 1 CFU in mTSB+n enrichments could be detected with all five methods, while the threshold for detection in BPW enrichments was 1 CFU for the Dynabeads and 10 CFU for VIDAS-ICE, VIDAS-UP, GeneDisc, and the LightCycler. In similar studies, (Vernozy-Rozand et al, 1998) consistently detected E. coli O157 contamination from food samples containing 8 CFU/25 g with VIDAS-ICE after a 24 hr incubation, whilst Wright, Chapman, and Siddons (1994) …”

Section: Relative Sensitivity Of E Coli O157 Detection Methodsmentioning

confidence: 64%

Evaluation of test-kits for the detection of Escherichia coli O157 in raw meats and cattle faeces.

Nyati¹,

Heuvelink

Heerwaarden

et al. 2012

Int. J. Food Stud.

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Escherichia coli O157 detection limits in artificially contaminated beef and cattle faeces samples were determined using Dynabeads anti E. coli O157 immunomagnetic beads, VIDAS-UP, VIDAS-ICE, and real-time PCR (GeneDisc and LightCycler) systems. Dynabeads anti-E. coli O157 immunomagnetic separation (IMS) and the GeneDisc cycler were the most sensitive methods, and could detect an initial 1 CFU in 25g beef samples after 6h of incubation in modified tryptone soya broth with novobiocin (mTSB+n) or buffered peptone water (BPW). The VIDAS-UP method could detect an initial 10 CFU, while VIDAS-ICE and the LightCycler methods could only detect an initial 100 CFU. Higher detection rates were achieved with 18 hour incubations, where an initial 1 CFU in a 25g sample could be detected with all five methods. For cattle faeces enrichments, Dynabeads anti-E. coli O157 IMS could detect an initial 1 CFU after a 6 h incubation in mTSB+n, while the VIDAS-UP and VIDAS-ICE methods could detect an initial 10 CFU and both PCR methods could only detect an initial 100 CFU. Detection rates were lower in BPW, compared to mTSB+n, with thresholds of 100 CFU for VIDAS-ICE, VIDAS-UP and GeneDisc methods, and >100 CFU for the LightCycler method.

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Immunomagnetic separation as a sensitive method for isolatingEscherichia coliO157 from food samples

Cited by 181 publications

References 24 publications

Enzymatic Digestion for Improved Bacteria Separation from Leafy Green Vegetables

Enzymatic Digestion for Improved Bacteria Separation from Leafy Green Vegetables

Immunomagnetic separation of the intestinal spirochaetes Brachyspira pilosicoli and Brachyspira hyodysenteriae from porcine faeces

Evaluation of test-kits for the detection of Escherichia coli O157 in raw meats and cattle faeces.

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