Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing

Abstract

The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The gram-positive bacteria Listeria monocytogenes, while not isolated as frequent as S. Typhimurium and E. coli, causes severe illness with an overall mortality rate of 30%. Thus, it is important that any outbreak of this pathogen is detected early and a fast trace to the source can be performed. In view of this, we have used the information provided by two fully sequenced L. monocytogenes strains to develop a MLVA assay coupled with high-resolution capillary electrophoresis and compared it to pulsed-field gel electrophoresis (PFGE) in two sets of isolates, one Norwegian (79 isolates) and one Swedish (61 isolates) set. The MLVA assay could resolve all of the L. monocytogenes serotypes tested, and was slightly more discriminatory than PFGE for the Norwegian isolates (28 MLVA profiles and 24 PFGE profiles) and opposite for the Swedish isolates (42 MLVA profiles and 43 PFGE profiles).

Introduction

The genus Listeria includes 6 different species and two subspecies where Listeria monocytogenes (L. monocytogenes) is the only species consistently associated with human illness. L. monocytogenes is a ubiquitous organism that is well adapted to persistence in the environment and has been isolated from soil, ground water, silage and decaying vegetation (Czuprynski, 2005, Hamon et al., 2006, Gandhi and Chikindas, 2007). L. monocytogenes is, however, most known for causing severe infection in animals and man (Portnoy et al., 2002, Czuprynski, 2005, Hamon et al., 2006, Gandhi and Chikindas, 2007). L. monocytogenes infections have been associated with drinking unpasteurised milk, soft cheeses, ice cream, raw vegetables, fermented raw-meat sausages, raw and cooked poultry, raw meats (all types), cold cuts, and raw and smoked fish, and its ability to grow at temperatures as low as 0.5 °C permits multiplication in refrigerated foods (Junttila et al., 1988, Czuprynski, 2005, Hamon et al., 2006, Gandhi and Chikindas, 2007). The disease, listeriosis, is clinically defined when the organism is isolated from blood, cerebrospinal fluid, or an otherwise normally sterile site. The symptoms of listeriosis include meningoencephalitis, septicaemia, abortions and febrile gastroenteritis (Czuprynski, 2005, Hamon et al., 2006, Gandhi and Chikindas, 2007). An average of 18.7 cases of human listeriosis was reported per annum in Norway between 2000 and 2006 to the Norwegian surveillance system for communicable diseases (MSIS: http://www.msis.no). An average of 47.6 cases of human listeriosis was reported per annum in Sweden from 2000 to 2006 (http://www.smittskyddsinstitutet.se/). Despite this relatively low number of cases, the severity of disease caused by L. monocytogenes makes it necessary to have a method that can rapidly detect outbreaks and be used in source identification and tracing. Several methods have been used to type L. monocytogenes isolates including serotyping (Allerberger, 2003), restriction enzyme analysis (REA) (Gerner-Smidt et al., 1996), ribotyping (Nocera et al., 1993), amplified fragment length polymorphism (AFLP) (Aarts et al., 1999), pulsed-field gel electrophoresis of macrorestricted DNA fragments (PFGE) (Heir et al., 2004, Martin et al., 2006). Recently multiple-locus variable-number tandem-repeats analysis (MLVA) with agarose-gel separation and multi-virulence-locus sequence typing (MVLST) has been described with good results (Murphy et al., 2007, Chen et al., 2007). We have designed a 5 loci MLVA method that benefits from the increased resolution power and speed of capillary electrophoresis instruments and compared the results with PFGE as this method is known to be highly discriminatory and reproducible for L. monocytogenes typing. MLVA is gaining acceptance as robust and rapid method for genotyping bacterial isolates (Lindstedt, 2005, Hyytia-Trees et al., 2006, van Belkum, 2007).