This study is a first step in the development of multilocus sequence typing (MLST) method for Listeria monocytogenes. Nine housekeeping genes were analyzed in a set of 62 strains isolated from different sources and geographic locations in Spain. These strains were previously characterized by pulsed-field gel electrophoresis (PFGE). Because of low diversity, two loci were discarded from the study. The sequence analysis of the seven remaining genes showed 29 different allelic combinations, with 22 of them represented by only one strain. The results of this sequence analysis were generally consistent with those of PFGE. Because MLST allows the easy comparison and exchange of results obtained in different laboratories, the future application of this new molecular method could be a useful tool for the listeriosis surveillance systems that will allow the identification and distribution of analysis of L. monocytogenes clones in the environment.Listeria monocytogenes is an opportunistic pathogen widely distributed in the environment. The ubiquity of this microorganism makes especially necessary the use of typing methods for the study of its epidemiology. Numerous molecular methods have been applied to the characterization of L. monocytogenes isolates, e.g., multilocus enzyme electrophoresis (MLEE) (1), pulsed-field gel electrophoresis (PFGE) (2, 29), random amplified polymorphic differences (19), and ribotyping (30), etc. By these methods the species is divided into two genetic divisions which are correlated with the flagellar antigen groups division I, composed of strains of serotypes 1/2a and 1/2c, and division II, which is composed of strains of serotypes 1/2b and 4b. Both divisions are characterized by nonoverlapping allelic variants of different genetic markers, suggesting strong linkage disequilibrium and an apparent lack of gene exchange between them (15). This is consistent with the hypothesis that the genetic structure of L. monocytogenes populations is basically clonal (21). Recently, an additional division has been proposed based on the variability of the sequence of several genes involved in virulence (22).MLEE has been the most widely used molecular method to study the genetic structure and epidemiology of pathogenic bacterial species. Recently, a novel molecular typing method based on the principles of MLEE (25) has been developed, multilocus sequence typing (MLST). This technique was primarily designed and validated for Neisseria meningitidis (17). Afterwards, it was successful in the characterization of several other pathogenic bacteria, such as Streptococcus pneumoniae (5), Streptococcus pyogenes (8), Staphylococcus aureus (7), and Campylobacter jejuni (4).MLST makes use of automated DNA sequencing to characterize the alleles present at different housekeeping genes.Because it is based on nucleotide sequence, it is highly discriminatory and provides unambiguous results that are directly comparable among laboratories via the internet (7). In addition, this method is particularly suited to global epidemiol...
A mass immunization campaign for 18-month to 19-year-olds was undertaken in Spain in 1996–1997 because of an epidemic of serogroup C meningococcal disease associated with a C:2b:P1.2,5 strain belonging to the A4 lineage. Surveillance for the “capsule-switching” phenomenon producing B:2b:P1.2,5 isolates was undertaken. Of 2,975 meningococci characterized, B:2b:P1.2,5 and B:2b:P1.2 antigenic combinations were found in 18 isolates; 15 meningococci were defined as serogroup B belonging to the A4 lineage.
Previous studies have shown that there is considerable variation in the methods and media used to determine the susceptibility of Neisseria meningitidis to antimicrobial agents in different countries. In this study, national and regional reference laboratories used a standardized methodology to determine the MICs of antibiotics used in the management of meningococcal infection. Fourteen laboratories participated in the study, determining the susceptibility to penicillin G, rifampin, cefotaxime, ceftriaxone, ciprofloxacin, and ofloxacin of a collection of 17 meningococci, of which 11 strains were previously defined as having intermediate resistance to penicillin (Pen I ) by sequencing and restriction fragment length polymorphism analysis of the penA gene. The MIC was determined by agar dilution and Etest with Mueller-Hinton agar (MH), MH supplemented with sheep blood (MH؉B), and MH supplemented with heated (chocolated) blood. Several laboratories encountered problems obtaining confluent growth with unsupplemented MH. MH؉B was considered to give the most congruent and reproducible results among the study laboratories. The modal MIC for MH؉B for each antibiotic and method was calculated to define the MIC consensus, allowing assessment of each individual laboratory's data in relation to the others. The agreement in each antibiotic/method/medium combination was defined as the percentage of laboratories with a result within one dilution of the modal result. For the whole study, an agreement of 90.6% was observed between agar dilution and Etest methods. The agreement in each laboratory/antibiotic/method combination ranged from 98.2% to 69.7%, with six laboratories demonstrating agreement higher than 90% and 11 more than 80%. The ability of the laboratories to detect the Pen I isolates ranged from 18.2% to 100%. The apparent difficulty in interpreting susceptibility to rifampin, particularly with the Etest method, is very interesting.
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