Immunomagnetic Separation Beads Research Articles

Gestational Diabetes Mellitus in Pregnancy Increased Erythropoietin Level Affecting Differentiation Potency of Haematopoietic Stem Cell of Umbilical Cord Blood.

Background: The in utero environment has many factors that can support cell differentiation. Cytokines, chemokines and growth factors play big roles in haematopoietic mechanisms. Some diseases like gestational diabetes mellitus (GDM) might affect the environment and haematopoietic stem cell (HSC) quality. The aim of this study is to investigate the adverse effects of GDM on umbilical cord blood (UCB) HSC in terms of differentiation potency including the UCB parameters used for banking and transplantation purposes.Methods: UCB-HSC was collected from 42 GDM and 38 normal pregnancies. UCB-HSC was isolated and further enriched using immuno-magnetic separation beads (MACS). The UCB-HSC were cultured in methylcellulose media to investigate the differentiation potency. The level of erythropoietin (EPO) and insulin in the UCB plasma was measured using enzyme linked immunoassay (ELISA) technique.Result: The UCB parameters; volume, total nucleated count (TNC) and total CD34+ cells were significantly reduced in the GDM group compared to the control group. The number of HSC progenitors' colonies were significantly reduced in the GDM group except for progenitor BFU-E, which was significantly increased (GDM = 94.19 ± 6.21, Control = 73.61 ± 2.73, p = 0.010). This data was associated with higher EPO level in GDM group. However, the insulin level in the GDM group was comparable to the Control group.Conclusion: Our results suggest that the changes in the in utero environment due to abnormalities during pregnancy such as GDM might affect the differentiation potency of UCB-HSC. These findings can be considered as an additional parameter for the inclusion and exclusion criteria for UCB banking, particularly for mothers with GDM.

Does each bead count? A reduced-cost approach for recovering waterborne protozoa from challenge water using immunomagnetic separation.

Giardia duodenalis and Cryptosporidium spp. are two of the most prominent aetiological agents of waterborne diseases. Therefore, efficient and affordable methodologies for identifying and quantifying these parasites in water are increasingly necessary. USEPA Method 1623.1 is a widely used and validated protocol for detecting these parasites in water samples. It consists of a concentration step, followed by parasite purification and visualization by immunofluorescence microscopy. Although efficient, this method has a high cost particularly due to the immunomagnetic separation (IMS) step, which is most needed with complex and highly contaminated samples. Based on this, the present study aimed to determine whether it is possible to maintain the efficiency of Method 1623.1 while reducing the amount of beads per reaction, using as a matrix the challenge water recommended by the World Health Organization. As for Giardia cysts, a satisfactory recovery efficiency (RE) was obtained using 50% less IMS beads. This was evaluated both with a commercial cyst suspension (56.1% recovery) and an analytical quality assessment (47.5% recovery). Although RE rates obtained for Cryptosporidium parvum did not meet Method 1623.1 criteria in any of the experimental conditions tested, results presented in this paper indicated the relevance of the described adaptations, even in challenge water.

Stability optimization of microbial surface-enhanced Raman spectroscopy detection with immunomagnetic separation beads

Immunomagnetic separation (IMS) beads with antibody coating are an interesting option for biosensing applications for the identification of biomolecules and biological cells, such as bacteria. The paramagnetic properties of the beads can be utilized with optical sensing by migrating and accumulating the beads and the bound analytes toward the focus depth of the detection system by an external magnetic field. The stability of microbial detection with IMS beads was studied by combining a flexible, inexpensive, and mass producible surface-enhanced Raman spectroscopy (SERS) platform with gold nanoparticle detection and antibody recognition by the IMS beads. Listeria innocua ATCC 33090 was used as a model sample and the effect of the IMS beads on the detected Raman signal was studied. The IMS beads were deposited into a hydrophobic sample well and accumulated toward the detection plane by a neodymium magnet. For the first time, it was shown that the spatial stability of the detection could be improved up to 35% by using IMS bead capture and sample well placing. The effect of a neodymium magnet under the SERS chip improved the temporal detection and significantly reduced the necessary time for sample stabilization for advanced laboratory testing.

Pooling of Immunomagnetic Separation Beads Does Not Affect Detection Sensitivity of Six Major Serogroups of Shiga Toxin–Producing Escherichia coli in Cattle Feces

Shiga toxin-producing Escherichia coli (STEC) of the serogroups O26, O45, O103, O111, O121, and O145, often called non-O157 STEC, are foodborne pathogens. Cattle are asymptomatic reservoirs for STEC; the organisms reside in the hindgut and are shed in the feces, which serve as the source of food product contaminations. Culture-based detection of non-O157 STEC involves an immunomagnetic separation (IMS) step to capture the specific serogroups in complex matrices, such as feces. The IMS procedure is time consuming and labor intensive because of the need to subject each fecal sample to six individual beads. Therefore, our objective was to evaluate whether pooling of IMS beads affects sensitivity of non-O157 STEC detection compared with using individual IMS beads. The evaluation was done by comparing detection of serogroups in feces spiked with pure cultures (experiments 1 and 2) and from feces (n = 384) of naturally shedding cattle (experiment 3). In spiked fecal samples, detection with pools of three, four, six, or seven beads was similar to, or at times higher than, detection with individual IMS beads. In experiment 3, the proportions of fecal samples that tested positive for the six serogroups as detected by individual or pooled beads were similar. Based on noninferiority tests, detection with pooled beads was not substantially inferior to detection with individual beads (P > 0.05). In conclusion, the pooling of IMS beads is a better option for detection of STEC serogroups in fecal samples compared with individual beads because the procedure saves time and labor and has the prospect of a higher throughput.

Comparison of subtypes of Listeria monocytogenes isolates from naturally contaminated watershed samples with and without a selective secondary enrichment.

Two enrichment methods for Listeria monocytogenes using Immuno Magnetic Separation (IMS) were tested to determine if they selected the same subtypes of isolates. Both methods used a non-selective primary enrichment and one included subculture in Fraser Broth, while the other involved direct plating of IMS beads. Sixty-two naturally contaminated watershed samples from the Central California Coast were used as a source of L. monocytogenes, and subtype diversity was measured by serotype and Multiple Number Variable Tandem Repeat Analysis (MLVA). Three different serotypes were detected from both methods with serotype 4b strains making up 87% of the isolates, serotype 1/2a making up 8%, and serotype 1/2b making up 5%. The data suggest that serotype 1/2a strains were more likely to be isolated from the Fraser Broth culture method. Sixty-two different MLVA types were detected and the more common MLVA types were detected by both culture methods. Forty-three MLVA types were detected only from one culture method or the other, while 19 types were detected from both culture methods. The most common MLVA type-12 was detected in 33 of the 62 water samples, and represented 31% of the isolates from both culture methods. This limited study provides evidence that using both enrichment culture methods allowed for detection of a greater diversity of isolates among the samples than the use of one method alone, and that a wide diversity of L. monocytogenes strains exist in this watershed.

Detection and Isolation of Low Levels of E. coli O157:H7 in Cilantro by Real‐Time PCR, Immunomagnetic Separation, and Cultural Methods with and without an Acid Treatment

Leafy greens such as cilantro, contaminated with Escherichia coli O157:H7, have been implicated in cases of human illnesses. High levels of microflora in fresh cilantro make recovery of low numbers of E. coli O157:H7 difficult. To improve upon current methods, immunomagnetic separation (IMS) techniques in combination with real-time PCR (RTiPCR) and selective enrichment protocols were examined. Rinsates were prepared from cilantro samples inoculated with low (~0.02 CFU/g) and slightly higher (~0.05 CFU/g) levels of E. coli O157:H7. Rinsate portions were enriched in modified buffered peptone water with pyruvate (mBPWp) for 5 h at 37 °C. After 5 h, selective agents were added to samples and further incubated at 42 °C overnight. Detection and recovery were attempted at 5 and 24 h with and without IMS. IMS beads were screened by RTiPCR for simultaneous detection of stx1, stx2, and uidA SNP. Additionally, broth cultures and IMS beads were streaked onto selective agar plates (Rainbow(®) agar, R&F(®) E. coli O157 Chromogenic medium, TC-SMAC and CHROMagar™ 0157) for isolation of E. coli O157:H7. Both broth cultures and IMS beads were also acid treated in Trypticase Soy Broth pH 2 prior to plating to selective media to improve upon cultural recovery. Although E. coli O157 strains were detected in most samples by PCR after 5 h enrichment, cultural recovery was poor. However, after 24 h enrichment, both PCR and cultural recovery were improved. Acidification of the broths and the IMS beads prior to plating greatly improved recovery from 24 h enrichment broths by suppressing the growth of competing microorganisms. Detection and recovery of Escherichia coli O157:H7 in fresh produce matrices (e.g., cilantro) can be complicated by high background microflora present in these foods. Rapid detection by molecular methods combined with effective enrichment and isolation procedures such as using immunomagnetic separation (IMS) techniques can quickly identify potential hazards to public health. Additional techniques such as acidification of enrichment broths can exploit acid resistance characteristics of pathogens such as E. coli O157:H7, facilitating their isolation in complex food matrices.

Efficacy of a post enrichment acid treatment for isolation of Escherichia coli O157:H7 from alfalfa sprouts

The enrichment, detection and isolation procedure in the current US FDA BAM have been shown effective for Escherichia coli O157:H7 in a wide variety of foods. Recently reported modifications to the enrichment protocol, including post-enrichment immunomagnetic separation (IMS) procedures have improved sensitivity of the method for alfalfa sprouts. However, cultural isolation on selective agar plates still presents a challenge in this food matrix.The focus of this study was to reduce levels of competing microflora and enhance isolation of E. coli O157:H7 on selective agars. We optimized the use of a short acid treatment after enrichment and with post-enrichment IMS beads. The optimized acid treatments were then evaluated in alfalfa sprouts artificially contaminated with E. coli O157:H7. After 5h enrichment of alfalfa sprouts contaminated at 0.2cfu/g, there was significant improvement in isolation on the selective plates following acid treatment of two types of IMS beads. Following 24h enrichment of alfalfa sprouts contaminated at low levels, E. coli O157:H7 was only recovered from 8/25 samples when no IMS or acid treatments were used. The use of only the acid treatment improved recovery to 19/25 samples. Following IMS of the enrichment broths, acid treatment increased isolation to 23/25 for Pathatrix™ and 24/25 for BeadRetriever™ IMS.Acid treatment (pH 2) of the enrichment broth for 30min or IMS beads for 7min is a simple and rapid way to greatly improve isolation of E. coli O157 from alfalfa sprout enrichments by reducing the interfering microflora on the selective media.

Detection by Multiplex Real-Time Polymerase Chain Reaction Assays and Isolation of Shiga Toxin–ProducingEscherichia coliSerogroups O26, O45, O103, O111, O121, and O145 in Ground Beef

Six Shiga toxin-producing Escherichia coli (STEC) serogroups, which include O26, O45, O103, O111, O121, and O145, are responsible for the majority of non-O157 STEC infections in the United States, representing a growing public health concern. Cattle and other ruminants are reservoirs for these pathogens; thus, food of bovine origin may be a vehicle for infection with non-O157 STEC. Methods for detection of these pathogens in animal reservoirs and in food are needed to determine their prevalence and to develop intervention strategies. This study describes a method for detection of non-O157 STEC in ground beef, consisting of enrichment in modified tryptic soy broth at 42°C, followed by real-time multiplex polymerase chain reaction (PCR) assays targeting stx(1), stx(2), and genes in the O-antigen gene clusters of the six serogroups, [corrected] and then immunomagnetic separation (IMS) followed by plating onto Rainbow® Agar O157 and PCR assays for confirmation of isolates. All ground beef samples artificially inoculated with 1-2 and 10-20 CFU/25 g of ground beef consistently gave positive results for all of the target genes, including the internal amplification control using the multiplex real-time PCR assays after enrichment in modified tryptic soy broth for a total of 24 h (6 h at 37°C and 18 h at 42°C). The detection limit of the real-time multiplex PCR assays was ∼50 CFU per PCR. IMS for O26, O103, O111, and O145 was performed with commercially available magnetic beads, and the IMS beads for O45 and O121 were prepared using polyclonal antiserum against these serogroups. A large percentage of the presumptive colonies of each serogroup picked from Rainbow Agar O157 were confirmed as the respective serogroups; however, the percent recovery of STEC O111 was somewhat lower than that of the other serogroups. This work provides a method for detection and isolation in ground beef and potentially other foods of non-O157 STEC of major public health concern.

Inoculation of beef with low concentrations of Escherichia coli O157:H7 and examination of factors that interfere with its detection by culture isolation and rapid methods.

Currently used industry testing programs require the ability to detect Escherichia coli O157:H7 in samples of beef trim or ground beef at levels as low as 1 CFU/375 g. We present a reliable protocol for generating a control inoculum for verification testing at this low concentration and evaluate its use. Results show that half of all samples received no cells when 1 CFU was the target concentration and that targets greater than 3 CFU were much more reliable. Detection by culture isolation and two commercial assays, Qualicon BAX-MP and BioControl GDS, detected 94% ± 11%, 92% ± 10%, and 92% ± 7% of samples inoculated with 5.4 CFU (range 1 to 9 CFU), respectively. We also examined the effect of background aerobic plate count (APC) bacteria and fat content effects on the detection of E. coli O157:H7. At APC concentrations below 6 log CFU/g, the rapid methods detected all beef trim samples inoculated with 26 CFU of E. coli O157:H7 per 65 g. At an APC of 6.7 log CFU/g, culture, BAX-MP, and GDS detected 100, 75, and 13%, respectively, of inoculated samples. Neither commercial method detected E. coli O157:H7 in the samples when APC was 7.7 log CFU/g, whereas culture was able to detect 63% of E. coli O157:H7 in the samples when APC was at this concentration. Increased fat content correlated with decreasing recovery of immunomagnetic separation beads, but this was not observed to interfere with detection of E. coli O157:H7.

Occurrence of Escherichia coli O157, O111 and O26 in raw ewe's milk and performance of two enrichment broths and two plating media used for its assessment

The occurrence of Escherichia coli O157, O111 and O26 in 159 raw ewe's milk samples was examined. Sample-aliquots were incubated simultaneously in TSB added with yeast extract (YETSB) and mTSB with novobiocin (N-mTSB). Serogroup-specific immunomagnetic separation (IMS) was then used and IMS beads were plated in a cefixime tellurite (CT)-containing media (CT-SMAC, CT-SBMAC and CT-RMAC for E. coli O157, O111 and O26, respectively) and E. coli O157:H7 chromogenic ID agar. A sweep of confluent growth from each medium was examined for the presence of E. coli O157 and O111 using PCR, and for E. coli O26 using a latex agglutination test. Enumeration of E. coli O157 and O111 was performed in the samples tested positive for the correspondent serogroup using the most probable number (MPN) method combined with PCR. Percentage occurrences of E. coli O157, O111 and O26 were 18.2, 8.2 and 5.7, respectively. Mean E. coli O157 and O111 levels were 0.22 and < 0.04 MPN/mL, respectively. Enrichment in YETSB resulted in higher detection rates of E. coli O157 and O26 than in N-mTSB. When YETSB was used as enrichment broth and for these last two serogroups, the analysis of the confluent growth from the CT-media gave more positive results than that from E. coli O157:H7-ID medium.

Prevalence of EnterohemorrhagicEscherichia coliSerotypes in Australian Beef Cattle

Beef cattle are known reservoirs of Escherichia coli O157; therefore, it is possible that they may be reservoirs for other enterohemorrhagic E. coli (EHEC) serotypes. This study investigated the prevalence of EHEC serotypes O26, O45, O91, O103, O111, O121, O145, and O157 in 300 beef cattle fecal samples. A combination of 300 (140 grain-fed and 160 grass-fed) cattle fecal samples were collected post-evisceration from Australian abattoirs. Enriched samples were tested for the putative virulence markers stx₁, stx₂, and eae using real-time PCR. Samples that tested positive for stx and eae were then tested for the target serotypes. Isolation was performed on any sample testing positive to stx and eae, and a target serotype using immunomagnetic separation for serotypes O26, O103, O111, O145, and O157 or colony hybridization for serotypes where immunomagnetic separation beads were not commercially available (O45, O91, and O121). Resulting isolates were characterized for the presence of stx₁, stx₂, eae, and ehxA using a multiplex PCR. Seventy-eight of the 300 samples (26%) were shown to contain stx and eae with 30 of these subsequently testing positive for the presence of at least one EHEC serotype. Of the 27 E. coli of EHEC serotypes isolated during the study, only 1 E. coli O91, 1 E. coli O26, and 5 E. coli O157 tested positive for the presence of any EHEC virulence markers. This study found that the overall prevalence of EHEC in Australian beef cattle is very low. Testing for the presence of virulence determinants and O-specific genes alone will overestimate the presence of pathogenic serotypes in beef. Isolation of strains of interest and confirming the presence of virulence determinants in those strains should be an essential part of any test protocol.

Effect of the enrichment time and immunomagnetic separation on the detection of Shiga toxin-producing Escherichia coli O26, O103, O111, O145 and sorbitol positive O157 from artificially inoculated cattle faeces

The purpose of this study was to evaluate the effect of enrichment time and immunomagnetic separation (IMS) on the efficiency of a method to isolate non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes O26, O103, O111, O145 and sorbitol positive (s+) O157 from artificially inoculated cattle faeces. Cattle faecal samples were inoculated with varying amounts (20–90 cfu/25 g and 200–900 cfu/25 g) of clinical STEC strains (including serotypes O26, O103, O111, O145 and O157 (s− and s+)), and recovered using a selective enrichment step of either 6 or 24 h, followed by plating on selective agars either preceded by IMS or not. Two types of IMS beads were used (Dynabeads ® and Captivate™ beads), and evaluated on pure broth suspensions of STEC. The 6-h enrichment instead of 24-h, had no significant effect on the recovery rate, only for serotype O145 a positive trend was observed. Using direct plating after enrichment, recovery rates were influenced by the inoculated serotype and the inoculation level. Using IMS (Dynabeads ® or Captivate™ beads), recovery of serotype O157 (s− and s+) was significantly improved, whereas for O26, O103, O111, and O145 no significant effect was observed. Results of IMS performed on pure suspensions of STEC, explained these serotype-depended findings in faeces; loss-making factors in the IMS procedure of some beads and the influence of the type of bead were shown. The method combining both enrichment periods with direct plating and IMS followed by plating, yielded a detection limit of 20–90 cfu/25 g. But, if only certain serotypes have to be investigated, the protocol can be simplified.

Modification to EPA Method 1623 to address a unique seasonal matrix effect encountered in some U.S. source waters

U.S. Environmental Protection Agency (EPA) Method 1623 is designed to detect and determine concentrations of Cryptosporidium oocysts and Giardia cysts in water through concentration, immuno-magnetic separation (IMS), and immuno-fluorescence assay with microscopic examination. A seasonal interference with the method was observed in some municipal source waters collected from reservoirs and as reported to Shaw Environmental, Inc. in the summers of 2005, 2006, and 2007. This interference, which was not confined to a single region of the nation, caused clumping of the IMS beads during the acid dissociation of the IMS procedure in Method 1623. This effect lowered method recoveries for both Cryptosporidium and Giardia; however, the effect was more pronounced for Giardia. A heat dissociation technique (Ware et al., (2003) J. Microbiol. Methods 55, 575–583) was shown to be a viable option for samples which demonstrate the clumping matrix effect and improved Giardia recoveries in partially clumped samples. The heat dissociation application holds promise for fully clumped samples and warrants further investigation.

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 °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 °C for 16 h in E.C. broth+novobiocin reaching numbers of (log 107.82–8.70). 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 109.6–7.5 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 106.4), 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.