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Two studies of meningococcal carriage state were carried out in Galicia (Spain) before and after a mass vaccination campaign between December 1996 and January 1997 against Neisseria meningitidis serogroup C with meningococcal serogroups A and C polysaccharide vaccine. The studies covered two areas with different incidence rates of meningococcal disease in 1996 (high and low incidence). Carriage rates of serogroup C showed a decrease in both areas, 47 and 65 % respectively, before and after the vaccination. Results showed a decrease in carrier state in the age groups 10-14- and 15-19-year-olds, but not in the 5-9-year-olds. These results demonstrate the effect of immunization on the reduction of the carriage state.

Background: Opsonophagocytosis and complement-mediated Neisseria meningitidis killing after vaccination were investigated., Methods: Twelve seronegative healthy volunteers received one dose of polysaccharide A/C vaccine and were followed for 3 years. Ex vivo serum killing rates with polymorphonuclear cells (PMN) and/or complement were performed at 0, 1.5, 6, 12, 18, 24, 30 and 36 months., Results: High mean total and median bactericidal antibodies were detected over time in all subjects. Considerable reduction of the initial inoculum was obtained only in the presence of complement, with or without PMN (with significant differences compared to curves without complement) a long time after vaccination., Conclusion: PMN did not increase post-vaccination bacterial killing, suggesting that antibody complement-mediated killing, and not opsonophagocytosis, is the main immune effector of the vaccine protection against N. meningitidis.

The increase in antibiotic resistance and the possible changes in serotype prevalence as a consequence of a new conjugated vaccine have contributed to renewed interest in the study of pneumococcal serotypes and their antibiotic resistances. Spain still has one of the highest penicillin resistance rates, but in the past 4-5 years a slight decrease has been observed. The level of resistance has not increased either, 12.7% of the 11 165 isolates studied showed high-level penicillin resistance but 94% of these had an MIC of only 2 mg/L. Serotypes 6, 9, 14, 19 and 23 included 83% of the penicillin-resistant pneumococci; the remaining 17% belonged to 18 different serotypes. We analysed these minor penicillin-resistant serotypes in view of their potential increase following a possible child vaccination programme. Four of these serotypes (11, 15, 21 and 35) were the most prevalent, and among them serotype 15 was particularly frequent with >50% of its strains resistant. The effective control of these minor penicillin-resistant serotypes should be based on continuous surveillance of pneumococcal epidemiology.

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.

Objective: To determine the incidence, as well as the implicated serotypes and patterns of antibiotic resistance of Streptococcus pneumoniae meningitis in Spanish children., Material and Method: We performed a prospective, multicenter study in five Autonomous Communities (Catalonia, Galicia, Madrid, Navarre and the Basque Country) for 1 year (1 February 2000 31 January 2001). All children aged 0-14 years with pneumococcal meningitis from all the hospitals in the Autonomous Communities studied were included. Diagnosis was based on clinical findings and isolation of S. pneumoniae in the cerebrospinal fluid/blood using routine methods or polymerase chain reaction. Serotyping was performed using the guellung reaction and/or immunoblotting and susceptibility to antibiotics was evaluated by the technique of agar dilution. The pediatric population aged 0-14 years in the Autonomous Communities studied comprises 2,290,304 children., Results: Fifty-two cases were identified. One patient was aged less than 2 months old, 25 (48 %) were aged 2-12 months, and 12 patients (23 %) were aged 12-24 months. The annual incidence per 100,000 children aged between 1 and 2 years was 17.75 cases (95 % CI: 11.59 26.01) and 8.39 cases (95 % CI: 4.67 15.79) respectively, and that for children in the first 2 and 5 years of life was 13.13 (95 % CI: 9.29 18.02) and 6.29 (95 % CI: 4.57 8.,45) cases respectively. Nearly half the strains isolated (47.6 %) showed reduced sensitivity to penicillin. The most frequent serotype was 19F (12 cases [28.6 %]). Eighty percent of the isolated serotypes were included in the formula of the heptavalent conjugate vaccine., Conclusions: The incidence of pneumococcal meningitis in children from five Spanish Autonomous Communities is high, nearly twice that found in a prior retrospective studied performed in the same population 1-3 years previously. Almost all the isolated serotypes were included in the heptavalent conjugate vaccine. Half the strains showed reduced sensitivity to penicillin.

We analysed a strain collection representative of the overall Neisseria meningitidis population circulating in an open community (46,000 inhabitants, Spain) during an endemic period (30 isolates from patients and 191 from throat cultures of healthy individuals) by both phenotypic and molecular techniques. Almost all patient isolates were assigned to three hyper-virulent lineages (ET-5 complex, ET-37 complex and cluster A4) by both multilocus enzyme electrophoresis (MEE) and pulsed-field gel electrophoresis (PFGE). In contrast, MEE and PFGE assigned 20% and 15% respectively of carrier isolates to the hyper-virulent clones (4% for both methods together). There was also a higher correlation between PFGE and phenotypes associated with virulent clones. These notable differences between the two molecular methods were further observed in more than half the carrier isolates, suggesting that the associations between these strains were distorted by recombination events. However, almost one-third of total endemic strains from symptom-free carriers and almost all patient strains belonged to clones defined by MEE and PFGE, with no known epidemiological connection. These data indicate low transmission and a weak clonal structure for N. meningitidis.

Unlabelled: This study analyses the serogroups/types (SGTs) and resistance to penicillin and erythromycin of 3921 strains isolated from 1990 to 1999 in children aged 0-14 y in Spanish hospitals of all the autonomous communities. Based on the age of the children, strains have been divided into five groups: 0-6 mo, > 6-1 y, > 1-2 y, > 2-5 y and > 5 y. While only eight SGTs were responsible for 80% of the infections in children from 6 mo to 2 y of age, this number increased to 11 and 16 for the groups > 2-5 y and > 5-14 y, respectively. SGTs 6, 14 and 19 were prevalent in blood and otic exudates. SGTs 1, 4, 5, 12 and 18 were more frequent in invasive disease but serotype 3 was clearly associated with otitis. Serotypes I and 5 were quite significant in children of over 2 y of age, and this should be taken into account in future vaccine formulations., Conclusion: Although high, the rate of penicillin resistance in the paediatric population has remained stable in recent years. Conversely, erythromycin resistance is still increasing in our country. Coverage by the 7-valent vaccine was 78 and 81% for blood and otic isolates, respectively. These coverage levels would be increased by 9% and 3% if 9-valent (plus 1 + 5 serotypes) were used and by an additional 2.6% and 7.6% using the 11-valent (plus 3 + 7) formulation.

A rise in the incidence of meningococcal disease has occurred in Spain in recent years, especially in some regions in the north-west of the country. Most cases have been caused by meningococci characterised as Neisseria meningitidis C:2b:P1.2,5. A total of 107 C:2b:P1.2,5 meningococcal isolates (60 from patients and 47 from carriers) and 12 isolates showing related antigenic combinations (C:2b:NST, C:2b:P1.2, C:2b:P1.5, C:NT:P1.2,5) was analysed by pulsed-field gel electrophoresis to determine the genetic variability of the epidemic and related strains. Endonucleases BglII and NheI were used to cut chromosomal DNA. When BglII was used, most of the C:2b:P1.2,5 isolates showed the same pulsotype regardless of whether they were from clinical cases or carriers. Isolates showing the principal profile after digestion with endonuclease BglII were analysed with NheI. Four pulsotypes were identified, of which two were found in only one isolate each. The major profiles (1 and 2) showed differential distribution among clinical and carrier isolates; pulsotype 1 was the most frequent among clinical isolates. However, the proportions of isolates showing profiles 1 and 2 were similar among carrier isolates. This could indicate that there are two variants of the C:2b:P1.2,5 strain with differing pathogenicity.

The in vitro activity of gemifloxacin versus those of 11 other antimicrobial agents against 400 strains of Neisseria gonorrhoeae was determined by microdilution with supplemented GC agar. A total of 37.5% of the strains were beta-lactamase positive. A total of 70 and 6.4% of the beta-lactamase-negative strains exhibited intermediate and high-level penicillin resistance, respectively. Ceftriaxone and gemifloxacin were the most active drugs (MICs at which 90% of isolates are inhibited, 0.01 versus 0.007 microg/ml, respectively), with 100% of strains inhibited by 0.12 microg/ml.

Background: To determine the anti-meningococcal C immunological activity by adding functional tests (opsonophagocytosis) to the classical serology techniques., Subjects and Methods: 42 adult volunteers were screened using serological methods (determination of total and bactericidal antibodies). Seronegative subjects were tested by opsonophagocytosis., Results: 24 subjects (57%) showed serological evidence of previous contact with Neisseria meningitidis group C antigens: 19 subjects had both total and bactericidal antibodies, two subjects had only total antibodies and three subjects had only bactericidal antibodies. Of the 18 seronegative subjects, five showed ex vivo activity in killing curves with or without polymorphonuclear cells: two subjects exhibited only complement-mediated bactericidal activity, one subject only opsonophagocytosis, and two subjects exhibited both activities., Conclusion: The addition of functional tests to the classical serological determination increases the evidence of previous contact with N. meningitidis antigens by 12%.

Background: In 1997, 18.5% of the cases of Meningococcal Disease caused b serogroup C in Andalusia were children between 2 and 4 years of age; ages where the initial immune response and the duration of the capsular A + C meningococcal polysaccharide vaccine is less than in older age groups. Research was designed in order to measure the immune response produced by this vaccine in children from 2 to 6 years of age and to compare it with the natural immunity present in unvaccinated children., Methods: I. Dual monitoring study: a) groups of children vaccinated previously and control groups, b) groups of children who were going to be vaccinated, for pre and post-vaccination (1, 6 and 12 months) analysis and a control group. II. The bactericidal activity was measured according to the standardised protocol of the CDC with regard to the strain of N. meningitidis C-11. The sera with bactericidal activity (TAB) > 1:8 were considered to be protective., Results: 1 and 2 months following vaccination, the proportion of TAB > 1:8 was significantly higher than that of the control group (65.6% and 73% in comparison to 2.2% and 12%). In the pre-vaccine and post-vaccine (after 6, 7, 12 and 13 months) verification, no significant difference between vaccinated individuals and controls was observed., Conclusions: The differences between vaccinated and unvaccinated individuals 1 and 2 months following vaccination indicate seroconversion in the vaccinated individuals. For the age group of between 2 to 6 years of age, the bactericidal activity acquired decline quickly, as, after 6 months, differences between this group and the control group are no longer observed.

In Galicia, Spain, a dramatic increase in the incidence of meningococcal disease was seen in the 1995-6. The annual incidence rose to 11 per 10(5) inhabitants, and 80% of identified strains were C:2b:P1.2,5. This led to the implementation of an intensive A+C vaccination campaign for the population aged 18 months to 19 years. During this campaign the prevalence of carriage in areas with high and low incidence was studied. Nasopharyngeal swabs were taken from 9796 subjects immediately before the administration of meningococcal vaccine, plated onto Thayer-Martin plates, incubated and sent for analysis to the Reference Laboratory for Neisseria in Spain. The prevalence of the C:2b: P1.2,5 strains was 0.6% (95% CI 0.29-0.88) in the high incidence area, and 0.41% (95 % CI 0.00-1.04) in the low incidence area, and that of serogroup C (all strains) 1.36% (95% CI 0.80-1.80) and 0.89% (95% CI 0.09-1.69) respectively. The prevalence of N. meningitidis (all strains) was almost the same in both areas (8%). Carriers of the epidemic strain were not found in the 2-4 year age group, that most affected by the disease. Our data showed a wide distribution but a low carriage rate of the epidemic strain C:2b:P1.2,5 in the high and low disease incidence areas studied; the difference in the carriage rates between the two areas was not statistically significant.

Multiply-antibiotic-resistant isolates of serogroup 19 Streptococcus pneumoniae, possessing altered penicillin-binding protein (PBP) 1A, 2B, and 2X genes that are indistinguishable from those of the Spanish multiresistant serogroup 23F clone, are now commonly encountered in Spain. Those isolates that have been serotyped express type 19F capsular polysaccharide. Serotyping of further isolates, and hybridization using a serotype 19F-specific probe, has shown that some of them are serotype 19A, rather than 19F. The Spanish multiresistant serotype 19A, 19F, and 23F multiresistant strains were all shown to be very closely related in overall genotype, as they were indistinguishable by REP-PCR and by the sequencing of internal fragments of three house-keeping genes. The serotype 19A multiresistant strains, like the serotype 19F multiresistant strains, therefore appear to be a serotype variant of the Spanish multiresistant serotype 23F clone, which presumably has arisen by recombination at the capsular locus.

Serogroup B has been the main cause of meningococcal disease in Spain since at least 1979, but in recent years an increase in the prevalence of infection due to serogroup C meningococci has been detected. In 1996, for the first time, most cases of meningococcal disease were caused by serogroup C strains. The sero/subtype of all serogroup C meningococci received from 1993 to June 1996 was determined, and the results showed that C:2b:P1.2,5, the most common phenotype in 1995 and 1996 (63% and 65%, respectively), represented only 4.8% of strains in 1993. The C:2b: P1.2,5 epidemic strains appear to be responsible for the high prevalence of serogroup C in Spain. One hundred fifty-one randomly selected serogroup C strains were analyzed by multilocus enzyme electrophoresis, ribotyping, and pulsed-field gel electrophoresis. Pulsed-field gel electrophoresis provided the most accurate information: more than 80% of the C:2b:P1.2,5 and C:2b:P1.2 isolates exhibited one of two very closely related profiles, while most of the C:2b:NST and C:2b:P1.5 strains had a pattern located at a genetic distance of 0.24 from those two profiles. The results show that C:2b:P1.2,5 strains represent a subclone or a genetic variant of the previously identified Spanish epidemic clone C:2b:non-subtypable strains.

Background: The aim of the study was to analyse meningococcal strains isolated from patients in Caracas (Venezuela) with epidemiological markers and to determine their susceptibility to antimicrobial agents., Methods: We analyzed 29 meningococcal clinical strains isolated during 1994 in Caracas by serogrouping, serotyping and subserotyping, multilocus enzyme analysis (MEE), ribotyping and pulse field electrophoresis (PFGE) profile. We also determined the Minimal Inhibitory Concentration (MIC) to 5 antimicrobial agents., Results: Twenty four (82.7%) were group C meningococcal strains. All group C meningococci were characterized as C: 2b: P1.5, belonging to the same electrophoretic type (ET) by MEE and showing the same profile by PFGE by using Bg/II endonuclease restriction enzyme. These group C meningococci showed two different patterns by ribotyping, with only one band difference. All Group C and one group B N. meningitidis isolates were moderately resistant to penicillin (MIC > or = 0.12 mg/l)., Conclusions: During 1994 an unusual high incidence of meningococcal strains moderately resistant to penicillin (PenMR) was detected in Caracas (Venezuela). A clone of C: 2b: P1.5 meningococci seem to be responsable for this high incidence of PenMR isolates.

We analysed a collection of 95 multiply antibiotic-resistant pneumococci, recovered since 1988 from 14 Spanish hospitals, that have MICs > or = 0.25 microgram benzylpenicillin ml-1. The majority of the isolates were of serogroups 14, 23, 6, 19 and 15, which are currently the serogroups mainly associated with multiresistance in Spain. All of the serogroup 23 isolates were members of the major Spanish serotype 23F multiresistant clone. Similarly, most of the serogroup 6 isolates were members of the major multiresistant serotype 6B clone, or variants of this clone. Eighteen of the 24 isolates of serogroup 19 were members of a highly penicillin-resistant clone that appears to be a serotype 19F variant of the major Spanish serotype 23F multiresistant clone. Eighteen of the 25 isolates of serotype 14 were members of a previously uncharacterized highly penicillin-resistant clone. Thirteen of the 16 isolates of serogroup 15 were members of a single previously unreported clone of serotype 15F that had moderate levels of resistance to penicillin. Approximately 65% of the multiresistant pneumococci that are currently circulating in Spain were members of the three new clones of serotype 14, 15F and 19F that we describe here, or the previously described serotype 6B and 23F clones. The other 35% of isolates were minor variants of the major clones, unrelated minor clones, and unique isolates, many of which appeared to have arisen by horizontal gene transfer events.

A vaginal isolate of Neisseria has been reported to resemble Neisseria meningitidis in biochemical characteristics but to react with serological reagents that are specific to the PI porin from Neisseria gonorrhoeae. We have confirmed that this isolate has the biochemical attributes of a meningococcus and have shown that it clusters among meningococcal isolates on a dendrogram based on isoenzyme variation within housekeeping enzymes from populations of N. meningitidis and N. gonorrhoeae. Furthermore, the sequences of the fbp and adk genes were typical of those of N. meningitidis and were distinct from those of N. gonorrhoeae. However, the porB gene was very similar to the por genes of N. gonorrhoeae isolates that express the PIB class of outer-membrane porin (differing from one gonococcal por allele at only a single nucleotide site), and was clearly distinct from the porB genes of N. meningitidis. The isolate therefore appears to be a typical meningococcus, except that its porB gene has been replaced with the por gene from a gonococcus.

Serogroup B Neisseria meningitidis is the main cause of meningococcal disease in Spain, but in recent years we have detected an increase in the prevalence of infection due to serogroup C meningococci. At the same time, the frequency of moderately penicillin-resistant (PenR) clinical isolates, which include greater numbers of serogroup C meningococci than do penicillin-susceptible (PenS) strains, has also been increasing. When we analyzed the prevalence of serogroups B and C in PenR and PenS meningococcal strains, we found a simultaneous increase in serogroup C strains and a decrease in serogroup B meningococci affecting both PenR and PenS isolates. To analyze this epidemiological change in Spain, we have applied serotyping, subtyping, and multilocus enzyme electrophoresis to serogroup C (PenR and PenS) strains. The two major serotypes were 2b and 2a in both groups (PenR and PenS), but our results suggested an association between serotype 2b and PenR strains. However, multilocus enzyme electrophoresis showed that 75% of the major serotypes belonged to the same electrophoretic type. It does not appear that a new clone distinct from those already established is contributing to the increase in serogroup C meningococci in Spain.

Penicillin-resistant (penr) clinical isolates of Neisseria meningitidis, which do not produce beta-lactamase, were first identified in Spain in 1985; the frequency of their recovery, which has been increasing in the past few years, reached 20% in 1989. Serogrouping, determination of serotypes and subtypes, and multilocus enzyme electrophoresis of the penr strains showed an extensive diversity. Resistance is due, at least in part, to a decreased affinity of penicillin-binding protein (PBP) 2 for penicillin. Similar low-affinity forms of PBP 2 are also found in penr isolates of Neisseria lactamica, Neisseria polysaccharea, and Neisseria gonorrhoeae. Genetic transformation of an N. meningitidis type strain to low-level penicillin resistance with DNA from resistant meningococci and other Neisseria species resulted in transformants that possessed low-affinity forms of PBP 2. These altered forms of PBP 2 have been shown to arise from recombinational events that replace parts of the PBP 2 gene with the corresponding regions from the PBP 2 genes of commensal Neisseria species.

Eighty-nine women prostitutes who underwent clinical and microbiologic examination were found to have gonococcal infection. The median age was 22; 92.1% were from urban areas. Nearly all the women prostitutes refrained from barrier methods (92.1%) and had contact with several partners (91.0%). The most frequent clinical findings were leukorrhea (50.6%), cervicitis (20.2%), and pelvic inflammatory disease (PID) (18.0%). Eighty-one women prostitutes (93.1%) had experienced a previous STD, with Chlamydia trachomatis (34.8%), Trichomonas vaginalis (30.3%), Neisseria gonorrhoeae (29.2%), and Ureaplasma urealyticum (23.6%) as the most frequent microorganisms isolated. Microorganisms associated with N. gonorrhoeae were isolated, mainly T. vaginalis (40.4%), C. trachomatis (31.5%), and Mycoplasma hominis (21.3%). For N. gonorrhoeae, the most frequent auxotypes were prototrophic (67.4%) and Proline (Pro)-dependent (14.6%); 2.2% of the strains were non-auxotypable. Beta-lactamase production was detected in three strains (3.4%) belonging to the auxotype/serovar: Lys/IA, Prototrophic/IB, and Pro/IB. The two former produced the 3.2-MDa "African" plasmid; the latter produced two plasmids (the 4.5-MDa "Asian" and the 24.5-MDa transfer plasmid.

Hospital infections caused by strains of Staphylococcus aureus non-typable (NT) by phages have occurred in three Spanish hospitals since 1981. Reverse typing allowed characterization of the strains in all three cases.

Between April 1983 and December 1985, 576 strains of Neisseria gonorrhoeae were isolated in our laboratory from patients attending Sexually Transmitted Diseases (STD) clinics. Of these, 61 (10.6%) were penicillinase-producing. Studies on these strains by plasmid analysis, auxotyping and serogrouping showed that the predominant type strains harboured the Asian resistance plasmid, were prototrophic, and were of serogroup W II/W III. About half of the strains, both of the African and Asian type, harboured the transfer plasmid. Strains of serogroup W II/W III were less sensitive to tetracycline and cefoxitin than serogroup W I strains.