Rapid and sensitive isolation of Campylobacter jejuni using immunomagnetic separation from patient specimens exposed to oxygen.

Abstract

This study describes a method for detecting Campylobacter jejuni in patient stools with subsequent isolation using antibody-magnetic beads in conjunction with selective culture and PCR. Monoclonal antibodies specific for the flagellin A and major outer membrane protein of C. jejuni were generated; two clones (1C7 and 4B2) were used to coat magnetic beads for immunomagnetic separation (IMS). C. jejuni strain NCTC11168 was recovered from human stool samples spiked with varying concentrations (10¹-10⁵ CFU/mL) by Campylobacter (Campy)-IMS or a conventional culture-based method and plated on modified charcoal-cefoperazone-deoxycholate agar; the number of colonies was enumerated. The detection limits of Campy-IMS and conventional culture-based method with spiked stool samples were 10² and 10⁴ CFU/mL, respectively. The sensitivity of IMS-PCR was 10-10,000-fold higher than that of direct PCR. The recovery rate of C. jejuni from spiked stools stored for 12 to 72 h decreased from 72.3 to 5.9% with Campy-IMS and from 48.5 to 0.1% with the conventional culture-based method. Importantly, of 20 PCR (+)/bacterial culture (-) samples that were diagnosed as probable cases according to general criteria, 95% (19/20) were confirmed positive by Campy-IMS. Thus, this study suggests a solution to overcome the problems caused by the inconsistency between probable and confirmed cases of Campylobacter infection.

Importance: The isolation, cultivation, and maintenance of Campylobacter spp. are difficult because of the microaerophilic conditions and specific medium needed. Although selective media are useful for the initial isolation of Campylobacter, subsequent exposure of the sample to oxygen has a detrimental effect on the positive culture rate of Campylobacter, significantly lowering the isolation rate from patient samples. In this study, the detection limit was improved by combining immunomagnetic separation and PCR methods to quickly detect Campylobacter jejuni in clinical patient stool samples using antibody-magnetic beads. Therefore, this study is expected to improve confirmation of C. jejuni infection where diagnosis would previously fail with patient samples because of oxygen exposure, inappropriate diagnostic methods, and interference from other bacteria in the sample.