Rapid Detection and Isolation of Escherichia coli O104:H4 from Milk Using Monoclonal Antibody-coated Magnetic Beads.

Mirella Luciani,Fabio Minelli,Elisabetta Di Giannatale,Laura Manna,Manuela Tittarelli,Katiuscia Zilli,Tiziana Di Febo,Gisella Armillotta,Alfredo Caprioli

doi:10.3389/fmicb.2016.00942

Abstract

Monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) of Escherichia coli O104:H4 were produced by fusion of Sp2/O-Ag-14 mouse myeloma cells with spleen cells of Balb/c mice, immunized with heat-inactivated and sonicated E. coli O104:H4 bacterial cells. Four MAbs specific for the E. coli O104:H4 LPS (1E6G6, 1F4C9, 3G6G7, and 4G10D2) were characterized and evaluated for the use in a method for the detection of E. coli O104:H4 in milk samples that involves antibody conjugation to magnetic microbeads to reduce time and increase the efficiency of isolation. MAb 1E6G6 was selected and coupled to microbeads, then used for immuno-magnetic separation (IMS); the efficiency of the IMS method for E. coli O104:H4 isolation from milk was evaluated and compared to that of the EU RL VTEC conventional culture-based isolation procedure. Milk suspensions also containing other pathogenic bacteria that could potentially be found in milk (Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus) were also tested to evaluate the specificity of MAb-coated beads. Beads coated with MAb 1E6G6 showed a good ability to capture the E. coli O104:H4, even in milk samples contaminated with other bacteria, with a higher number of E. coli O104:H4 CFU reisolated in comparison with the official method (121 and 41 CFU, respectively, at 103 E. coli O104:H4 initial load; 19 and 6 CFU, respectively, at 102 E. coli O104:H4 initial load; 1 and 0 CFU, respectively, at 101 E. coli O104:H4 initial load). The specificity was 100%.

Highlights

Escherichia coli (Enterobacteriaceae) is a Gram-negative, facultative anaerobic bacterium that is commonly found in the lower intestinal tract of healthy animals and humans
Prior to 2011, STEC serogroup O104 was not considered as a major STEC serogroup, it had been associated with an outbreak of diarrhea in the US and with sporadic cases in European countries and Korea (European Centre for Disease Prevention and Control [ECDC] and European Food Safety Authority [EFSA], 2011; Baranzoni et al, 2014)
Characterization by western blotting revealed that the four selected Monoclonal antibodies (MAbs) reacted with the LPS of E. coli O104:H4; Figure 1 shows the ladder-like pattern of LPS bands with molecular weights between 60 and 15 kDa recognized by the MAbs

Summary

Introduction

Escherichia coli (Enterobacteriaceae) is a Gram-negative, facultative anaerobic bacterium that is commonly found in the lower intestinal tract of healthy animals and humans. Prior to 2011, STEC serogroup O104 was not considered as a major STEC serogroup, it had been associated with an outbreak of diarrhea in the US and with sporadic cases in European countries and Korea (European Centre for Disease Prevention and Control [ECDC] and European Food Safety Authority [EFSA], 2011; Baranzoni et al, 2014) The concern about this serogroup increased in May-July 2011, with the occurrence of two outbreaks of bloody diarrhea and HUS in Europe: one in Germany (around 4000 cases of bloody diarrhea, 850 cases of HUS and 50 deaths), and a much smaller outbreak in southwest France (15 cases of bloody diarrhea, 9 of which progressed to HUS). The specific combination of the higher adherence to intestinal cells, physical survival, Stx production and antibiotic resistance, shows the high genomic plasticity of E. coli O104:H4 and could explain the high virulence of the epidemic strain (Bielaszewska et al, 2011; Scheutz et al, 2011)

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Conclusion