Selective recovery of Streptosporangium fragile from soil by indirect immunomagnetic capture

Abstract

A polyclonal anti body raised to Stmptosporangium fkagi/e spores reacted strongly and specifically with the immunizing strain and to a number of related species of Streptosporangium, as determined by dot immunoblotting. An indirect immunomagnetic capture method was developed for the recovery of the target organism from sterile and non-sterile soil, using sheep anti-rabbit M-280 Dynabeads. The effects of different soil blocking agents, antibody labelling concentrations and spore/Dynabead capture times on the recovery of 5. fragile spores were investigated. Pre-blocking of antibody binding sites within the soil, with either 2% partially hydrolysed gelatin or 10% skimmed milk, was essential prior to immunomagnetic capture. Increasing the capture time from 15 to 60 min did not affect spore recovery; however, a 10-fold decline in the magnetic bead concentration did result in a significantly lower recovery of spores from soil. 5. fragi/e was selectively enriched (1 :190=fold) when present as a mixed population with Arthmbacter oxydans in sterile soil. The indirect immunomagnetic capture method was used to selectively recover 5. fragile spores seeded into non-sterile soil, although some background binding of non-target bacteria was noted. The target was successfully recovered from a sterile soil microcosm after 14 d incubation and the capture rate was increased by the inclusion of an initial soil dispersion and biomass concentration procedure, using the ion-exchange resin Chelex 100.Keywords : immunocapture, Streptosporangium, soil, magnetic beads, selective isolation INTRODUCTIONThere is an increasing need to improve methods for the detection and isolation of bacteria from environmental samples, both for the recovery of genetically engineered strains and microbial inoculants (Ford & Olsen, 1988;Morgan et al., 1989; Trevors e t al., 1993) and for the isolation of rare and unusual bacteria for use in industrial screening programmes (Nolan & Cross, 1988 ;Shearer, 1987).Immunomagnetic capture (IMC) represents a potentially important development within this field, allowing the specific recovery of target bacteria from a diverse range of environmental samples. The method relies upon the interaction between cell-surface antigens and antibodies which can be attached to magnetized polystyrene beads.Abbreviations: DIB, dot irnrnunoblotting; IMC, irnrnunornagnetic capture; MPC, magnetic partlcle concentrator; Non, Nonidet P-40.Separation of target cells from a suspension can be achieved using a magnetic field. IMC has been widely used in clinical studies for the separation or depletion of target cells from a mixed population (Gaudernack et ai., 1986; Gruhn e t al., 1991 ; Silvestri e t al., 1992). More recently it has been used to enrich for and enhance the detection of pathogenic bacteria present in contaminated foodstuffs (Johne e t al., 1989; Mansfield e t a!., 1993; Parmer eta/., 1992; Skjerve etai., 1990) and faeces (Luk & Lindberg, 1991 ; Tomoyasu, 1992). Work has also focused on the detection and isolation of bacteri...

Colorimetric detection of Shewanella oneidensis based on immunomagnetic capture and bacterial intrinsic peroxidase activity

“…In spite of high specificity to target cells in environmental samples29, reverse transcription fluorescent in situ hybridization, which utilizes sequence-specific nucleic acid labeled with fluorophores as probes, involves a very time-consuming and labor-intensive sample treatment procedure, including fixation, hybridization and washing. Fortunately, the recently developed IMC technique is not only simple and time-saving, but efficient in removing inhibitory substances and heterogeneous bacteria in samples3031. In addition, IMC is non-destructive for the target cells.…”

Immunomagnetic recovery of Mycobacterium bovis from naturally infected environmental samples

“…Previous IMC studies have been successful in the selective recovery of Streptomyces lividans (Wipat et al. 1994) and Streptosporangium fragile (Mullins et al. 1995).…”

Electrical stimulation of microbial PCB degradation in sediment

“…Individual congeners and respective concentrations were obtained from Mullin et al . (Mullin et al, 1984). Fifty-five additional congeners not present in the Aroclor 1232:1248:1262 mixture that were potential dechlorination products were added to the calibration table containing the Aroclor congeners.…”