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...

The phenotypic and genetic analysis results for 84 isolates ofLactococcus garvieae (including 62 strains from trout with lactococcosis from four different countries, 7 strains from cows and water buffalos with subclinical mastitis, 3 from water, and 10 from human clinical samples) are presented. There was great phenotypic heterogeneity (13 different biotypes) based on the acidification of saccharose, tagatose, mannitol, and cyclodextrin and the presence of the enzymes pyroglutamic acid arylamidase andN-acetyl-β-glucosaminidase. L. garvieaealso exhibited high genetic diversity by pulsed-field gel electrophoresis (PFGE), with 19 different pulsotypes among the isolates of L. garvieae studied. Only epidemiologically related strains, like the Spanish and Italian fish isolates and the cow and water buffalo isolates, displayed a close genetic relationship by PFGE, while the strains isolated from sporadic clinical cases, like the human isolates, were genetically unrelated. Overall, a general correlation between phenotypic and genetic data was observed. Epidemiological analysis of biotype and PFGE results indicated that the trout lactococcosis outbreaks in Spain and Portugal and those in France and Italy were produced by genetically unrelated clones. In Spain, two different clones were detected; the outbreaks diagnosed from 1995 onward were produced by a clone (biotype 2, pulsotype A1) which, although genetically related, was different from the one that was responsible for the outbreaks studied between 1991 and 1994 (biotype 1, pulsotype B). The Portuguese isolate had a biochemical profile identical to that of the Spanish strain isolated from 1995 onward and is also genetically closely related to this strain (pulsotype A2). There was a close relationship between the two pulsotypes (E and F) found in the Italian isolates. The French isolate (biotype 3, pulsotype D) was not genetically related to any other L. garvieaefish isolate. These results suggest the existence of diverse infection sources for the different lactococcosis outbreaks.

One Neisseria gonorrhoeae strains highly resistant to azithromycin AzHLR (MIC >2048 mg/L) was isolated in Argentina in 2001 and it has been characterized by N. gonorrhoeae multiantigen sequence typing (NG-MAST) as ST696, suggesting a different event to other isolates in Europe. Neither, mtrR mutations or presence of mef gene were detected.

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.