Sequencing of the porB gene: a step toward a true characterization of Neisseria meningitidis.

Variations in class 2/3 (PorB) proteins form the basis for meningococcal serotyping. Antibodies against these proteins are bactericidal, making serotyping results useful not only for epidemiological surveillance of meningococcal disease but also for identifying potential vaccine components. A total of 20 to 60% of meningococcal B and C isolates from any given population are nontypeable (NT) using a panel of monoclonal antibodies. To analyze the mechanisms responsible for the nonserotypeability characteristic in Neisseria meningitidis, we (i) established the nucleotide sequences of porB gene in 146 meningococcal strains (95 not recognized by the serotyping panel), (ii) identified 18 new allelic variants of the porB gene, (iii) correlated allelic variants with serotypes, (iv) suggest the nontypeability characteristic in those 95 NT strains, and (v) reject the possibility of variation in the levels of PorB expression.

Orogenital transmission of Neisseria meningitidis serogroup C confirmed by genotyping techniques.

Urethritis caused by Neisseria meningitidis in heterosexual patients is presumed to occur via orogenital contact, but confirmation has not been possible in most cases. Presented here is a case of urethritis caused by N. meningitidis, serogroup C, and the isolation of the same microorganism from the nasopharynx and endocervix of the patient's sexual partner. The similarity of the urethral and nasopharyngeal isolates' electrophoretic patterns, obtained using pulsed-field gel electrophoresis, proves the infection was transmitted via orogenital contact.

Impact of the meningococcal C conjugate vaccine in Spain: an epidemiological and microbiological decision.

The new meningococcal C conjugate vaccine became available in Spain and was included in the infant vaccination schedule in 2000. A catch-up campaign was carried out in children under six years of age. As a consequence, the incidence of meningococcal disease caused by serogroup C has fallen sharply during the last three epidemiological years in Spain. The risk of contracting serogroup C disease in 2002/2003 fell by 58% when compared with the season before the conjugate vaccine was introduced. There was also an important decrease in mortality. Three deaths due to serogroup C occurred in the age groups targeted for vaccination in 2002/2003, compared with 30 deaths in the same age groups in the season before the launch of the vaccine campaign. In the catch-up campaign the vaccine coverage reached values above 92%. For the 2001, 2002 and 2003 routine childhood immunisation programme coverage values ranged from 90% to 95%. During the past three years a total of 111 cases of serogroup C disease have been reported in patients in the vaccine target group. Most of the vaccination failures occurred during the epidemiological year 2002/2003. Eight (53%) vaccine failures occurred in children who had been routinely immunised in infancy, and could be related to a lost of protection with time since vaccination. The isolation of several B:2a:P1.5 strains (ST-11 lineage) is noteworthy. These may have their origin in C:2a:P1.5 strains which, after undergoing genetic recombination at the capsular operon level, express serogroup B. These strains could have relevant epidemic potential.

Impact of the meningococcal C conjugate vaccine in Spain: an epidemiological and microbiological decision.

The new meningococcal C conjugate vaccine became available in Spain and was included in the infant vaccination schedule in 2000. A catch-up campaign was carried out in children under six years of age. As a consequence, the incidence of meningococcal disease caused by serogroup C has fallen sharply during the last three epidemiological years in Spain. The risk of contracting serogroup C disease in 2002/2003 fell by 58% when compared with the season before the conjugate vaccine was introduced. There was also an important decrease in mortality. Three deaths due to serogroup C occurred in the age groups targeted for vaccination in 2002/2003, compared with 30 deaths in the same age groups in the season before the launch of the vaccine campaign. In the catch-up campaign the vaccine coverage reached values above 92%. For the 2001, 2002 and 2003 routine childhood immunisation programme coverage values ranged from 90% to 95%. During the past three years a total of 111 cases of serogroup C disease have been reported in patients in the vaccine target group. Most of the vaccination failures occurred during the epidemiological year 2002/2003. Eight (53%) vaccine failures occurred in children who had been routinely immunised in infancy, and could be related to a lost of protection with time since vaccination. The isolation of several B:2a:P1.5 strains (ST-11 lineage) is noteworthy. These may have their origin in C:2a:P1.5 strains which, after undergoing genetic recombination at the capsular operon level, express serogroup B. These strains could have relevant epidemic potential.

Molecular characterization of ciprofloxacin resistance of gonococcal strains in Spain.

BACKGROUND: Over the past several years, the emergence of gonococcal isolates with intermediate or full resistance to fluoroquinolones has become a significant concern in several countries, including Spain. GOAL: The goal was to determine the occurrence of ciprofloxacin resistance among Neisseria gonorrhoeae strains in Spain during 2000 to 2001 and determine the frequency and patterns of mutations at gyrA, gyrB, and parC genes in these isolates. STUDY DESIGN: Eleven ciprofloxacin-resistant strains (with MICs ranging from 1 to 64 micrograms/mL) and two intermediate isolates (with MICs of 0.12 and 0.5 microgram/mL) were found. Mutations were identified by polymerase chain reaction and direct sequencing of the amplified products. RESULTS AND CONCLUSIONS: Alterations at Ser-91 and Asp-95 in GyrA were detected in all strains except one, an isolate for which the MIC was 0.12 microgram/mL. Alterations in ParC were more variable, and there was no clear correlation between the number of parC mutations and the level of resistance. No alterations at gyrB gene associated with ciprofloxacin resistance were found. The resistance was distributed among different types of strains, suggesting that the increase in the incidence of ciprofloxacin-resistant strains in Spain was not exclusively due to the appearance of a single-strain outbreak.

Development of a multilocus sequence typing method for analysis of Listeria monocytogenes clones.

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.

Antibiotic resistance of Neisseria gonorrhoeae in Spain: trends over the last two decades.

In vitro activities of six antimicrobial agents against 2966 strains of Neisseria gonorrhoeae, isolated in Spain between 1983 and 2001, were determined. The percentages of intermediately susceptible and resistant isolates to penicillin (MIC > or = 0.12 mg/L) and tetracycline (MIC > or = 0.5 mg/L) were very high over the period of study. Strains intermediately susceptible to cefoxitin were identified at a variable percentage during the study. All N. gonorrhoeae isolates were susceptible to spectinomycin and ceftriaxone. Recently, resistance to ciprofloxacin has emerged.

The epidemic wave of meningococcal disease in Spain in 1996-1997: probably a consequence of strain displacement.

During 1996 and 1997 an epidemic wave of meningococcal disease took place in Spain. Initial studies described the antigenic expression of the epidemic strain as C:2b:P1.2,5 and proposed that it was a variant of the previously identified Spanish C:2b:non-subtypable epidemic strain. To clarify this hypothesis, 1036 C:2b:P1.2(5) and 76 C:2b:NST isolates obtained during 1992-1999 were analysed by pulsed-field gel electrophoresis. The majority of the C:2b:P1.2,5 and C:2b:P1.2 isolates showed one of two very closely related profiles. During the epidemic period, 80% of the C:2b:NST strains showed these two pulsotypes. However, before the epidemic wave, most of these C:2b:NST strains (60%) showed a profile that was found infrequently among C:2b:P1.2,5 and C:2b:P1.2 isolates. A similar evolution was observed in C:2b:P1.5 isolates. Thirty-four C:2b:P1.2(5) and 10 C:2b:NST isolates, exhibiting representative pulsotypes, were subjected to multi-locus sequence typing. Isolates belonging to both A4 and ET-37 lineages were identified. These data point to the possibility that the A4 cluster has displaced the ET-37 complex among serogroup C meningococci in Spain.

What about antibiotic resistance in Neisseria lactamica?

The in vitro activity of penicillin, ampicillin, cefotaxime, ceftriaxone, rifampicin and ciprofloxacin against 286 Neisseria lactamica isolates was determined by agar dilution and the category of susceptibility was analysed in accordance with the criteria used for Neisseria meningitidis. All isolates were considered to have intermediate susceptibility to penicillin. A total of 1.7% of the isolates were resistant to ampicillin but all were susceptible to cefotaxime and ceftriaxone. Rifampicin MICs ranged between 0.12 and 2 mg/L. Six isolates (2.1%) showed decreased susceptibility to ciprofloxacin.

Phenotypic and genetic characterization of Lactococcus garvieae isolated in Spain from lactococcosis outbreaks and comparison with isolates of other countries and sources.

The phenotypic and genetic analysis results for 84 isolates of Lactococcus 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 and N-acetyl-beta-glucosaminidase. L. garvieae also 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. garvieae fish isolate. These results suggest the existence of diverse infection sources for the different lactococcosis outbreaks.