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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.

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 105 inhabitants, and 80% of identified strains were C[ratio ]2b[ratio ]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[ratio ]2b[ratio ]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[ratio ]2b[ratio ]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.

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.

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 l...

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.

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.

Background: Classifying bacteria into species is problematic. Most microbiologists consider species to be groups of isolates that share some arbitrary degree of relatedness of biochemical or molecular (such as DNA sequence) features and that, ideally, are clearly delineated from all other groups of isolates. The main problem in applying to bacteria a biological concept of species based on the ability or inability of their genes to recombine, is that recombination appears to be rare in bacteria in nature, as indicated by the strong linkage disequilibrium between alleles found in most bacterial populations. However, there are some naturally transformable bacteria in which assortative recombination appears to be so frequent that alleles are in, or close to, linkage equilibrium. For these recombining populations a biological concept of species might be applicable. Results: Populations of Neisseria gonorrhoeae and Neisseria meningitidis from Spain were analysed by multilocus enzyme electrophoresis. The data indicate that assortative recombination occurs frequently within populations, but not between populations. Similarly, the sequences of two house-keeping genes show no evidence of intragenic recombination between N. gonorrhoeae and N. meningitidis . Conclusions: N. gonorrhoeae and N. meningitidis represent extremely closely related ‘sexual' populations that appear to be genetically isolated in nature, and thus conform to the biological concept of species. The extreme uniformity of N. gonorrhoeae house-keeping genes suggests that this species may have arisen recently as a clone of N. meningitidis that could colonize the genital tract. Ecological isolation — of populations that can colonize the genital tract from those that can colonize the nasopharynx — may have been an important component in speciation, leading to a lower frequency of recombination between species than within species.

The meningococcal NMB0035 locus encodes a 47 kDa outer-membrane protein that is highly conserved antigenically, and is able to induce antibodies during infection and bactericidal responses in vitro. This study analysed the surface exposure of this protein using specific antibodies in flow cytometry assays and determined its nucleotide sequence in 33 Neisseria strains. Genomic analyses revealed no significant differences in the nucleotide or amino acid sequences, but flow cytometry showed that surface accessibility was highly variable among the strains. These results suggest that masking by and/or association with lipo-oligosaccharides or other membrane molecules can be crucial for antigen accessibility, which must be thoroughly analysed in new vaccine candidates.

More than 50% of the nontypeable (NT) pneumococcal strains received in our laboratory for reference purposes are isolated in sporadic cases of conjunctivitis. To determine the genetic structure of the population of these NT conjunctival strains, we analyzed 75 pneumococci (40 NT and 35 typeable) isolated from conjunctivas and 30 (15 NT and 15 typeable) isolated from other sources. The NT and typeable conjunctival strains grouped in separate clusters, whereas NT and typeable pneumococci isolated from other sources were similarly distributed. NT conjunctival strains belonged to two well-differentiated clonal lineages. The first, represented by three newly described sequence types, featured fully antibiotic susceptible strains and appeared to be characteristic of conjunctival tissue; the second, represented by the previously described ST344, had a pattern of multiresistance to penicillin, tetracycline, and erythromycin and shared a genetic background with some NT strains isolated from other sources.

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.

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.

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 Neusseria 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%.

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 β-lactamase positive. A total of 70 and 6.4% of the β-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 μg/ml, respectively), with 100% of strains inhibited by 0.12 μg/ml.

SUMMARYHospital infections caused by strains ofStaphylococcus aureusnon-typable (NT) by phages have occurred in three Spanish hospitals since 1981. Reverse typing allowed characterization of the strains in all three cases.

SUMMARYBetween April 1983 and December 1985, 576 strains ofNeisseria gonorrhoeaewere 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 WII/WIII. About half of the strains, both of the African and Asian type, harboured the transfer plasmid. Strains of serogroup WII/WIII were less sensitive to tetracycline and cefoxitin than serogroup WI strains.

Summary. A scheme is proposed for biotyping Gardnerella vaginalis, based on detection of hippurate hydrolysis, 0-galactosidase (ONPG) and lipase, and fermentation of arabinose, galactose and xylose. Seventeen biotypes were found among 197 strains from asymptomatic women and patients with bacterial vaginosis (non-specific vaginitis). The distribution of biotypes was similar in both populations but some biotypes were found more frequently in patients. The proposed scheme is compared with those previously described.

We analysed a collection of 95 multiply antibiotic-resistant pneumococci, recovered since 1988 from 14 Spanish hospitals, that have MICsor = 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.