Yersinia enterocolitica recovery by immunomagnetic separation

LAZARTE OTERO VALERIA; LUCERO ESTRADA CECILIA STELLA MARYS; FAVIER GABRIELA ISABEL; GALLO GIOVANNA LUCRECIA; ESCUDERO MARÍA ESTHER; STEFANINI ANA MARÍA

Carlos Paz, Córdoba

Congreso; VI Congreso Argentino de Microbiología General; 2009

Sociedad Argentina de Microbiología General

Yersinia enterocolitica, a human enteropathogen, is transmitted through contaminated water and food. Its recovery by culture is difficult when a low number of this bacterium is found. The immunomagnetic separation (IMS) is a separation and concentration method which uses paramagnetic polystyrene particles (PMP) covered with specific antibodies against surface antigens of microorganisms under study. In the present work, IMS was performed for evaluating the Y. enterocolitica recovery from an enrichment broth. The local virulence plasmid bearing strains, Y. enterocolitica 2/O:9 and Y. enterocolitica 3/O:3 were grown in trypticase soy broth (TSB) at 22° C overnight. Their initial concentrations were standardized at OD600 0.2. Serial dilutions of each strain were performed and spread on trypticase soy agar (TSA), and countings of 2 x 108 CFU/ml for 2/O:9 and 1x 109 CFU/ml for 3/O:3 were obtained. One milliliter of each dilution was transferred to 9 ml of TSB and these bacterial suspensions were utilized on the same day (day 0) and 24 h after being incubated at 22° C (day 1). Dilutiones 10-2 to 10-5 were assayed on day 0 and dilutions 10-6 to 10-9 were assayed on day 1. Y. enterocolitica counting obtained without IMS treatment (wIMS) were compared to those obtained after IMS treatment (aIMS). The 2,8 μm diameter PMP covered with sheep anti rabbit-IgG (Dynalbeads) reacted with rabbit anti Y.enterocolitica 2/O:9 IgG and rabbit anti Y.enterocolitica 3/O:3 IgG and were ready for the challenge against suspensions of Y.enterocolitica O:9 and Y. enterocolitica O:3. The antibody-antigen reaction was performed at 35° C for 30 min with gentle agitation. The procedure was concluded by performing three washes with PBS-0.02% Tween 20 and final suspensions in 100 μl PBS. A measured volume of each Eppendorf tube was spread on Mac Conkey agar for estimating the Y. enterocolitica recovery after IMS. This counting was compared to that obtained before performing IMS. The best performance of IMS in the Y. enterocolitica recovery was observed for the highest bacterial dilutions (dilutions 10-6 on day 0 and 10-9 on day 1). Thus, counts of 80 CFU/ml (wIMS) and 1322 CFU/ml (aIMS) for the strain 2/O:9 and 70 CFU/ml (wIMS) and 1535 CFU/ml (aIMS) for the strain 3/O:3 were obtained on day 0. Also, counts of 4 x 104 CFU/ml (wIMS) and 6,3 x 104 CFU/ml (aIMS) for the strain 2/O:9 and < 200 CFU/ml (wIMS) and 6090 CFU/ml (aIMS) for the strain 3/O:3 were obtained on day 1. The Y. enterocolitica recovery was up to 30 times more effective by culture after IMS than by culture without IMS.