Separation of the bacterial species, Escherichia coli, from mixed-species microbial communities for transcriptome analysis

Abstract

BackgroundThe study of bacterial species interactions in a mixed-species community can be facilitated by transcriptome analysis of one species in the community using cDNA microarray technology. However, current applications of microarrays are mostly limited to single species studies. The purpose of this study is to develop a method to separate one species, Escherichia coli as an example, from mixed-species communities for transcriptome analysis.ResultsE. coli cells were separated from a dual-species (E. coli and Stenotrophomonas maltophilia) community using immuno-magnetic separation (IMS). High recovery rates of E. coli were achieved. The purity of E. coli cells was as high as 95.0% separated from suspended mixtures consisting of 1.1 - 71.3% E. coli, and as high as 96.0% separated from biofilms with 8.1% E. coli cells. Biofilms were pre-dispersed into single-cell suspensions. The reagent RNAlater (Ambion, Austin, TX) was used during biofilm dispersion and IMS to preserve the transcriptome of E. coli. A microarray study and quantitative PCR confirmed that very few E. coli genes (only about eight out of 4,289 ORFs) exhibited a significant change in expression during dispersion and separation, indicating that transcriptional profiles of E. coli were well preserved.ConclusionsA method based on immuno-magnetic separation (IMS) and application of RNAlater was developed to separate a bacterial species, E. coli as an example, from mixed-species communities while preserving its transcriptome. The method combined with cDNA microarray analysis should be very useful to study species interactions in mixed-species communities.

The Paradox of Mixed‐Species Biofilms in the Context of Food Safety

“…Continuous‐optimizing confocal reflection microscopy may also be used to monitor the time course of mixed‐species biofilms formations since this procedure does not necessitate a staining procedure (Inaba and others ). Recently developed molecular technologies such as DNA microarray (Manuzon and Wang ; Dai and others ; Pammi and others ), denaturing high‐performance liquid chromatography (Manuzon and Wang ), proteomics (reviewed in Di Cagno and others ; Klein and others ; Sánchez and others ), and in vivo studies in mice (Pammi and others ) and/or other animal models could also help to identify the gene regulation and virulence of mixed‐species biofilms in specific food niches (reviewed in Sauer ). The combination of cDNA microarray with an immunomagnetic separation technique has been useful for mixed‐species biofilms analysis (Dai and others ).…”

“…Recently developed molecular technologies such as DNA microarray (Manuzon and Wang ; Dai and others ; Pammi and others ), denaturing high‐performance liquid chromatography (Manuzon and Wang ), proteomics (reviewed in Di Cagno and others ; Klein and others ; Sánchez and others ), and in vivo studies in mice (Pammi and others ) and/or other animal models could also help to identify the gene regulation and virulence of mixed‐species biofilms in specific food niches (reviewed in Sauer ). The combination of cDNA microarray with an immunomagnetic separation technique has been useful for mixed‐species biofilms analysis (Dai and others ). A DNA array was successfully employed to demonstrate significant differences in gene expression in mixed‐species biofilms, compared to monospecies biofilms (Redanz and others ).…”

Simple and rapid sample preparation system for the molecular detection of antibiotic resistant pathogens in human urine

“…This approach can be easily incorporated into a passive sample preparation device, and requires no complex fluidics. Antibody coated beads attach to surface antigens of specific bacteria and can be used to separate them from a mixed community for detection by fluorescence microscopy (Wen et al 2013 ), PCR (Beyor et al 2009 ), loop mediated isothermal amplification (Wang et al 2011 ) or transcriptome studies (Dai et al 2011 ). Aptamers can also be used instead of antibodies as the capture molecule (Suh et al 2014 ).…”

“…Aptamers can also be used instead of antibodies as the capture molecule (Suh et al 2014 ). However, some of these approaches have significant limitations such as requiring the bacteria to be in a particular buffer to facilitate capture, or requiring a nucleic acid extraction after cell capture (Dai et al 2011 ; Suh et al 2014 ), in addition to the time and financial burden of producing the capture molecules. Furthermore, antibody approaches are limited to only a single known target organism, which has already been isolated and cultured, and is assumed to represent the wild-type strains.…”

Pathogen–nematode interaction: Nitrogen supply ofListeria monocytogenesduring growth inCaenorhabditis elegans

“…The debris was pelleted by centrifugation at 10 4 × g , and the supernatant was filtered (pore size 10 μm and 3 μm) to remove nematodal cells. The filtrate was pelleted (6 min, 6654 × g ), resuspended in 2 ml of PBS containing 0.5% biotin‐free albumin fraction V (Carl Roth, Karlsruhe, Germany), and used for subsequent immunomagnetic separation with the MACS cell separation system (Miltenyi Biotech, Auburn, CA) according to Dai and colleagues () using biotin‐conjugated anti‐ Listeria sp. antibody (ViroStat, Portland, ME).…”