tbpB Genes Research Articles

Antimicrobial resistance and molecular characteristics of Neisseria gonorrhoea isolates in Ghana.

Gonorrhoea is a disease associated with humans and caused by Neisseria gonorrhoea. N. gonorrhoea's ability to evolve and evade various treatment regimens can lead to untreatable gonorrhoea. In the absence of a viable vaccine and a national database on the antimicrobial resistance (AMR) and molecular characteristics of N. gonorrhoea, and with reliance on a syndromic management regime, continuous national antimicrobial resistance surveillance and molecular characterization of N. gonorrhoea remain imperative. Only two gonococcal studies have described N. gonorrhoea's molecular characteristics linked to AMR in Ghana. Secondary N. gonorrhoea isolates (n=4) were collected from two metropolises in Ghana: Tamale in the northern sector (n=1) and Accra in the southern sector (n=3). The isolates were confirmed and characterized using polymerase chain reaction (PCR) targeting the porB and tbpB genes, and the disc diffusion method was used to evaluate AMR. N. gonorrhoea multi-antigen sequence typing (NG-MAST) and porin B (porB) gene sequence analyses were employed to reveal the molecular epidemiology and evolutionary trajectory, respectively. All four isolates showed resistance to at least four of the tested antibiotics. One isolate showed resistance to all seven antibiotics, i.e. ceftriaxone, azithromycin, ciprofloxacin, tetracycline, erythromycin, togamycin and penicillin. NG-MAST typing revealed isolate S3 (MZ313864) as ST211. The locus of S2 (MZ313863) (transferrin-binding protein B; tbpB) was identified as tbpB1844, and its porB locus, as porB6412, with only 4 closely related variants but with 15 nucleotide differences. However, its sequence type does not exist. The porB analysis identified isolate S3 (MZ313864) to be found globally, while S2 (MZ313863) is unique to this study. Despite the small number of isolates tested, this study recorded multidrug resistance and previously unknown gonococcal variants based on porB gene. Additionally, the molecular typing schemes revealed a disparity between NG-MAST and the National Center for Biotechnology Information (NCBI) platforms. There is a need for continuous gonococcal AMR and molecular surveillance in Ghana to contribute to the global efforts to describe circulating strains and support proper application of the syndromic management regime to gonorrhoea.

Antimicrobial Resistance Profiling and Genome Analysis of the penA-60.001 Neisseria gonorrhoeae Clinical Isolates in China in 2021.

N. gonorrhoeae antimicrobial resistance (AMR) is an urgent public health threat. With dissemination of FC428-related clones, the efficacy of ceftriaxone has become controversial. Agar dilution and whole-genome-sequencing were used to analyze AMR. High-resistance to penicillin (75.2%), tetracycline (87.9%), ciprofloxacin (98.3%), ceftriaxone (8.9%), cefixime (14.3%) and azithromycin (8.6%) was observed among 463 isolates first collected in China in 2021. All penA-60.001 clones exhibited resistance to ceftriaxone or cefixime, and 1/12 of the cases were resistant to azithromycin. ngMAST and ngSTAR of penA-60.001 isolates showed SNPs in porB, tbpB, ponA, gyrA and parC genes were the major causes of different sequence types. MLST-7365 (n = 5) and MLST-1903 (n = 3) were main genotypes, and the other four strains featured MLST-10314, MLST-13871, MLST-7827 and MLST-1600. Furthermore, a set of resistance markers, such as penA, blaTEM-1 and blaTEM-135, and virus factors were detected. Most penA-60.001 strains were fully mixed with global FC428-related clones; 2021-A2 and F89 had the same origin; and 2021-A1 exhibited a unique evolutionary trajectory. Our results provide the first demonstration of extremely severe AMR rates of N.gonorrhoeae in China in 2021, particularly those strains with ceftriaxone decreased-susceptibility, and reveal the sustained transmission of penA-60.001 subclones might further threaten treatment effectiveness.

Genotype Diversity in the Local Arkhangelsk (Russia) Neisseria gonorrhoeae Population: Emergence Mechanisms and Relation with Antimicrobial Resistance

132 clinical isolates of N. gonorrhoeae collected in Arkhangelsk region during 2014–2016 were analyzed in accordance with the NG-MAST protocol (Neisseria gonorrhoeae Multi Antigen Sequence Typing). A diversity of the analyzed strains was represented by 53 NG-MAST sequence-types; 30 of them were new registered due to earlier unknown either porB and tbpB sequences or their combinations. Three pathways of the diversity formation are supposed: long-term circulation of some NG-MAST-types with sequential transmission via the “sexual chains” (40.1% of the total population); introduction of new molecular types (27.3%); appearance of point mutations in porB and tbpB genes with the emergence of previously unknown genotypic variants of N. gonorrhoeae (32.6%). Emerged NG-MAST types inherited the mutational profile in penA, ponA, rpsJ, gyrA, and parC genes followed by conservation of antimicrobial resistance in the local N. gonorrhoeae population, whereas these determinants do not play a significant role in contemporary gonococci molecular evolution.

NG-MAST GENOTYPING OF NEISSERIA GONORRHOEAE STRAINS ISOLATED IN RUSSIAN FEDERATION IN 2012 - 2015

Aim. Characteristics of contemporary strains of N. gonorrhoeae using NG-MAST genotyping (.Neisseria gonorrhoeae multi-antigen sequence typing), isolation of dominating variants and analysis of their territorial distribution. Materials and methods. 440 strains of N. gonorrhoeae isolated in 2012 - 2015 in 19 subjects of the Russian Federation were used in the study. Genotyping was carried out based on sequencing of variable regions of porB and tbpB genes. Identification of alleles and sequence-types was carried out according to http://www.ng-mast.net. Results. 172 NG-MAST genotypes were identified, 100 of which -were described for the first time. 807,1152, 1544,5714 and 5941 were predominating sequence-types, typical for several subjects of the Russian Federation and some neighboring countries, as well as sequence-types 8583 and 9476 described for the first time, that were exclusively distributed in the regions of their emergence. Sequence-types 225, 1407 and 2992, that are epidemically significant in distant countries, were represented by single isolates in this study. Conclusion. A peculiar and significant genetic diversity of N. gonorrhoeae strains, circulating in the Russian Federation, with characteristic bond of several NG-MAST genotypes to one or several regions, was shown.

Genetic characteristics of Neisseria gonorrhoeae isolates from Guangzhou city and relationship between their multiantigen sequence typing sequence types and ciprofloxacin resistance

Objective To investigate genetic characteristics of Neisseria gonorrhoeae (N. gonorrhoeae)isolates from Guangzhou city in 2014, and to analyze the relationship of N. gonorrhoeae multi-antigen sequence typing (NG-MAST)sequence types(STs)with ciprofloxacin resistance. Methods An agar dilution method was used to determine the minimal inhibitory concentration(MIC)of ciprofloxacin in 97 N. gonorrhoeae isolates from Guangzhou city. PCR was performed to amplify the gyrA, parC, porB and tbpB genes from these isolates, followed by gene sequencing and determination of NG-MAST STs. Results Of the 97 N. gonorrhoeae isolates, 95 (97.9%)were resistant to ciprofloxacin, including 41 high-level (MIC ≥ 16 mg/L)and 54 low-level (1 mg/L ≤ MIC < 16 mg/L)resistant strains. Mutations were detected at codons 91 and 95 encoding serine in the gyrA gene of all the 95 ciprofloxacinresistant strains, and in the parC gene of 93 resistant strains. The frequency of the mutation at codon 87 in the parC gene was 85.4% (35/41)in high-level resistant strains, significantly higher than that in low-level resistant strains (59.3%[32/54], χ2 = 7.64, P < 0.05). MAST STs were successfully determined for all the 97 N. gonorrhoeae isolates except 1 isolate with incorrect PCR amplicons. Of the 96 genotyped isolates, 50 were assigned to 35 known STs by using the NG-MAST website (www. ng-mast.net), among which, 10 STs each contained 2 to 4 isolates. The most common ST was ST5309. Phylogenetic tree analysis revealed that the 96 genotyped N. gonorrhoeae isolates could be classified into 2 groups, and the proportion of isolates with MIC ≥ 16 mg/L is 46.4% (39/84)in group 1, but only 1/12 in group 2 (χ2 = 6.27, P = 0.012). Conclusions High-level resistance of N. gonorrhoeae to ciprofloxacin may be mainly associated with the mutation at codon 87 in the parC gene. NG-MAST STs may be related to the degree of ciprofloxacin resistance. Key words: Neisseria gonorrhoeae; Ciprofloxacin; Drug resistance, bacterial; NG-MAST

Changes in the six most common sequence types of Neisseria gonorrhoeae, including ST4378, identified by surveillance of antimicrobial resistance in northern Taiwan from 2006 to 2013.

There has been no longitudinal study of drug susceptibility in Neisseria gonorrhoeae in Taiwan since 2006. We collected 1090 gonococcal isolates from Taipei City Hospital, Taiwan from April 2006 to August 2013. We used a disk diffusion assay to determine the susceptibility to five antibiotics and an E-test to determine the minimum inhibitory concentrations for cefixime and ceftriaxone in isolates with resistance. Neisseria gonorrhoeae-multi Antigen Sequence Typing and DNA sequencing of the por and tbpB genes were used to identify sequence types. Among the 1090 isolates, the resistances to penicillin, ciprofloxacin, cefpodoxime, cefixime, and ceftriaxone were 61.01%, 83.39%, 9.63%, 6.70%, and 2.39%, respectively. The highest minimum inhibitory concentrations of cefixime and ceftriaxone were 0.19mg/L and 0.50mg/L, respectively. There were 327 sequence types. The four most common sequence types in homosexuals were ST4378, ST359, ST4654, and ST547; the two most common sequence types in heterosexuals were ST421 and ST419. Each of these sequence types had more than 25 isolates. There were significant differences in the sequence types in patients with different sexual orientations (p<0.001). Oral cefixime or ceftriaxone injections were used as first-line drugs for the treatment of gonorrhea from 2006 to 2013 because gonorrhea isolates had low minimum inhibitory concentrations for these two drugs. The abrupt emergence of ST4378 (closely related to the notorious ST1407) since 2009 is a cause for alarm. Changes in sexual behavior, including an increase in sexual activity without the use of condoms, may have contributed to the peak in gonorrhea in 2010. Further molecular epidemiological investigations are required.

P2.030 Utility of Neisseria Gonorrhoea Positive APTIMA Specimens to Assess Strain Diversity and Antibiotic Resistance

BackgroundSamples collected from community patients and patients visiting the Sexually Transmitted Infection (STI) clinics, submitted for the detection of Neisseria gonorrhoea (NG) in Alberta, Canada, are tested by the FDA...

Evidence for a common gene pool and frequent recombinational exchange of the tbpBA operon in Mannheimia haemolytica, Mannheimia glucosida and Bibersteinia trehalosi

The tbpBA operon was sequenced in 42 representative isolates of Mannheimia haemolytica (32), Mannheimia glucosida (6) and Bibersteinia trehalosi (4). A total of 27 tbpB and 20 tbpA alleles were identified whilst the tbpBA operon was represented by 28 unique alleles that could be assigned to seven classes. There were 1566 (34.8% variation) polymorphic nucleotide sites and 482 (32.1% variation) variable inferred amino acid positions among the 42 tbpBA sequences. The tbpBA operons of serotype A2 M. haemolytica isolates are, with one exception, substantially more diverse than those of the other M. haemolytica serotypes and most likely have a different ancestral origin. The tbpBA phylogeny has been severely disrupted by numerous small- and large-scale intragenic recombination events. In addition, assortative (entire gene) recombination events, involving either the entire tbpBA operon or the individual tbpB and tbpA genes, have played a major role in shaping tbpBA structure and it's distribution in the three species. Our findings indicate that a common gene pool exists for tbpBA in M. haemolytica, M. glucosida and B. trehalosi. In particular, B. trehalosi, M. glucosida and ovine M. haemolytica isolates share a large portion of the tbpA gene, and this probably reflects selection for a conserved TbpA protein that provides effective iron uptake in sheep. Bovine and ovine serotype A2 lineages have very different tbpBA alleles. Bovine-like tbpBA alleles have been partially, or completely, replaced by ovine-like tbpBA alleles in ovine serotype A2 isolates, suggesting that different transferrin receptors are required by serotype A2 isolates for optimum iron uptake in cattle and sheep. Conversely, the tbpBA alleles of bovine-pathogenic serotype A1 and A6 isolates are very similar to those of closely related ovine isolates, suggesting a recent and common evolutionary origin.

Neisseria gonorrhoeae multi-antigen sequence typing: a new genotyping method

The typing of gonococcus involves phenotypic analysis and molecular genotyping. Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) is a sequence-based genotyping method with a broad prospect. NG-MAST system is not only suitable for cultured specimens, but also for swab samples. It allows the typing of N.gonorrhoeae strains by blasting against databases at NG-MAST Web site following amplification of variable internal regions of por and tbpB genes of gonococcal strains using PCR and sequencing of PCR products.Compared with other genotyping methods, NG-MAST appears simple and objective, and tends to produce results with satisfactory comparability between national and international laboratories. This system has been extensively used in predicting gonococcal resistance, analyzing sexual networks, identifying outbreak and epidemic strains, and so on. Key words: Neisseria gonorrhoeae ; Sequence analysis ; Evaluation studies ;

Neisseria gonorrhoeae multi-antigen sequence typing using non-cultured clinical specimens

ObjectivesThe Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) system, based on PCR amplification and sequence analysis of the gonococcal porB and tbpB genes, is widely used for molecular typing of gonococcal...

Distribution of transferrin binding protein B gene (tbpB) variants among Neisseria species

BackgroundTransferrin binding protein B (tbpB), an outer membrane lipoprotein, is required for the acquisition of iron from human transferrin. Two tbpB families have been documented in Neisseria meningitidis: an isotype I tbpB gene of 1.8 kb and an isotype II tbpB gene of 2.1 kb, the former expressed by meningococci in the disease-associated ST-11 clonal complex and the latter found among meningococci belonging to the hyper-invasive clonal complexes including ST-8, ST-18, ST-32, ST-41/44 as well as N. gonorrhoeae isolates. The origin of the isotype I tbpB gene is unknown, however several features in common with non-pathogenic Neisseria and the ST-11 clonal complex N. meningitidis isolate FAM18 have been documented leading to the hypothesis that the isotype I tbpB gene may also be shared between non-pathogenic Neisseria and ST-11 meningococci. As a result, the diversity of the tbpB gene was investigated in a defined collection of Neisseria species.ResultsTwo families of isotype I tbpB were identified: family A containing conserved genes belonging to ST-11 meningococci, N. polysaccharea and N. lactamica isolates and family B including more diverse isotype I tbpB genes from N. sicca, N. mucosa, N. flava, N. subflava as well as N. cinerea, N. flavescens and N. polysaccharea isolates. Three isotype II tbpB families were identified with: family C containing diverse tbpB genes belonging to N. polysaccharea, N. lactamica, N. gonorrhoeae and N. meningitidis isolates, family D including another subset of isotype II tbpB genes from N. lactamica isolates and family E solely composed of N. gonorrhoeae tbpB genes.ConclusionThis study reveals another instance of similarity between meningococci of the ST-11 clonal complex and non-pathogenic Neisseria with the origin of the isotype I tbpB gene resulting from a horizontal genetic transfer event occurring between these two populations.

Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) of ciprofloxacin resistant isolates of Pretoria, South Africa

Gonococcal isolates resistant to ciprofloxacin collected from 2004 to 2005, in the Pretoria region, were characterised using Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). The isolates were obtained from men presenting...

Characterization of Proline, Citrulline, and Uracil Auxotrophic Plasmid-Carrying Neisseria gonorrhoeae Strains in Canada, 1993–2003

Proline, citrulline, and uracil (PCU) Neisseria gonorrhoeae is a common auxotroph in Canada and resistance to antibiotics in this auxotroph is rare. The goal of this study was to determine the characteristics of the PCU strains that have plasmids. PCU strains sent between 1993 and 2003 for reference work to the National Microbiology Laboratory were analyzed for their plasmid profiles and antimicrobial susceptibilities. PCU strains containing plasmids and/or antimicrobial resistant were serotyped, and por and tbpB genes were sequenced to determine their antigenic types. Forty strains containing plasmids were identified; serotype IB-11 and por/tbpB sequence type 2 was the most common pattern (28 of 40 strains [70%]). One strain had plasmids that conferred resistance to penicillin and tetracycline and 4 plasmidless strains were resistant to tetracycline. PCU auxotrophic N. gonorrhoeae carrying plasmids and/or that have chromosomal genes mutated to resist antimicrobials have been identified in Canada. This shows the competency of this pathogen to survive under antimicrobial pressure and the importance of continuous surveillance.

Characterization of Neisseria meningitidis isolates from recent outbreaks in Ethiopia and comparison with those recovered during the epidemic of 1988 to 1989.

The objectives of this study were to collect and characterize epidemic meningococcal isolates from Ethiopia from 2002 to 2003 and to compare them to 21 strains recovered during the previous large epidemic of 1988 to 1989. Ninety-five patients in all age groups with clinical signs of meningitis and a turbid cerebrospinal fluid (CSF) sample were included in the study of isolates from 2002 to 2003. Seventy-one patients (74.7%) were confirmed as having Neisseria meningitidis either by culture (n = 40) or by porA PCR (n = 31) of their CSF. The overall case fatality rate (CFR) was 11.6%; the N. meningitidis-specific CFR was 4.2%. All 40 strains were fully susceptible to all antibiotics tested except sulfonamide, were serotyped as A:4/21:P1.20,9, and belonged to sequence type 7 (ST-7). The strains from 1988 to 1989 were also equally susceptible and were characterized as A:4/21:P1.20,9, but they belonged to ST-5. Antigenic characterization of the strains revealed differences in the repertoire of lipooligosaccharides and Opa proteins between the old and the recent strains. PCR analysis of the nine lgt genes revealed the presence of the lgtAHFG genes in both old and recent strains; lgtB was present in only some of the strains, but no correlation with sequence type was observed. Further analysis showed that in addition to their pgm alleles, the Ethiopian ST-5 and ST-7 strains also differed in their tbpB, opa, fetA, and lgtA genes. The occurrence of new antigenic structures in strains sharing the same serogroup, PorA, and PorB may help explain the replacement of ST-5 by ST-7 in the African meningitis belt.

The gonococcal Fur-regulated tbpA and tbpB genes are expressed during natural mucosal gonococcal infection.

Iron is limiting in the human host, and bacterial pathogens respond to this environment by regulating gene expression through the ferric uptake regulator protein (Fur). In vitro studies have demonstrated that Neisseria gonorrhoeae controls the expression of several critical genes through an iron- and Fur-mediated mechanism. While most in vitro experiments are designed to determine the response of N. gonorrhoeae to an exogenous iron concentration of zero, these organisms are unlikely to be exposed to such severe limitations of iron in vivo. To determine if N. gonorrhoeae expresses iron- and Fur-regulated genes in vivo during uncomplicated gonococcal infection, we examined gene expression profiles of specimens obtained from male subjects with urethral infections. RNA was isolated from urethral swab specimens and used as a template to amplify, by reverse transcriptase PCR (RT-PCR), gonococcal genes known to be regulated by iron and Fur (tbpA, tbpB, and fur). The constitutively expressed gonococcal rmp gene was used as a positive control. RT-PCR analysis indicated that gonorrhea-positive specimens where rmp expression was seen were also 93% (51/55) fbpA positive, 87% (48/55) tbpA positive, and 86% (14 of 16 tested) tbpB positive. In addition, we detected a fur transcript in 79% (37 of 47 tested) of positive specimens. We also measured increases in levels of immunoglobulin G antibody against TbpA (91%) and TbpB (73%) antigens in sera from infected male subjects compared to those in uninfected controls. A positive trend between tbpA gene expression and TbpA antibody levels in sera indicated a relationship between levels of gene expression and immune response in male subjects infected with gonorrhea for the first time. These results indicate that gonococcal iron- and Fur-regulated tbpA and tbpB genes are expressed in gonococcal infection and that male subjects with mucosal gonococcal infections exhibit antibodies to these proteins.

Detection and subtyping of Actinobacillus pleuropneumoniae strainsby PCR-RFLP analysis of the tbpA and tbpB genes

A PCR-based procedure for detection and serotype identification of Actinobacillus pleuropneumoniae strains was developed and evaluated. The A. pleuropneumoniae tbpA and tbpB genes were used as targets for amplification of DNA fragments, with a pair of specific primers for each gene. Amplification with tbpA primers rendered a 2.8-kb PCR product from all 12 A. pleuropneumoniae reference strains as well as from Actinobacillus suis strain CCM 5586, while amplification of a 1.9-kb PCR product was observed when testing ten Haemophilus parasuis strains of different serovars. Amplification of the tbpB gene from A. pleuropneumoniae serotypes 1, 6, 8 and 12, and A. suis CCM 5586 rendered an identical 1.8-kb fragment, while from A. pleuropneumoniae serotypes 2, 3, 4, 7, 9, 10 and 11, and H. parasuis strains it produced a 1.7-kb fragment. No PCR amplification product was observed when examining strains of 19 other swine pathogens or closely related species. The minimal detection limit for whole-cell A. pleuropneumoniae templates was between 5–50 and 3 × 10 2–3 × 10 3 CFU when tbpA and tbpB specific primers, respectively, were used. Restriction fragment length polymorphism (RFLP) analysis of the PCR-generated products rendered different patterns, easily allowing us to discriminate between A. pleuropneumoniae, H. parasuis and A. suis and, more importantly, to distinguish ten RFLP A. pleuropneumoniae groups (the highest discrimination reported so far for a PCR assay with A. pleuropneumoniae), in such a way that the only serotypes with profiles identical to each other were 4 to 11 and 7 to 9. Moreover, the PCR-RFLP analysis was assayed in 36 A. pleuropneumoniae field isolates and in porcine samples (lungs and nasal swabs from experimentally infected animals). In both cases the system proved to be very efficient in A. pleuropneumoniae identification and serotype discrimination.

Human antibody responses to A and C capsular polysaccharides, IgA1 protease and transferrin–binding protein complex stimulated by infection with Neisseria meningitidis of subgroup IV-1 or ET-37 complex

The protein sequences of the IgA1 protease, TbpA and TbpB proteins differ between meningococci representative of serogroup A, subgroup IV-1 from epidemic disease in The Gambia and serogroup C, ET-37 complex from endemic disease in Mali. The uniformity of restriction endonuclease sites was determined for the iga, tbpA and tbpB genes among strains of both clonal lineages. Rare isolates had acquired a variant tbpAB operon by horizontal genetic exchange but all other strains were uniform within each clonal lineage. The quantitative levels of IgG to capsular polysaccharide, IgA1 protease and TBP complex were measured in paired acute phase and convalescent phase sera from The Gambia and from Mali using antigens from the homologous clonal lineages. IgG levels to these antigens were also measured in paired sera from healthy Gambians who permanently carried meningococci in the nasopharynx or did not. The results showed that disease stimulated IgG to each antigen in Mali and to all but TBP complex in The Gambia. Similarly, higher levels of IgG were found in sera from permanent carriers than in sera from permanent non-carriers. Acute phase sera from Mali contained low levels of IgG to C capsular polysaccharide (geometric mean value of 0.3 μg ml −1) while such sera from The Gambia contained higher and potentially protective levels of IgG to A polysaccharide (geometric mean of 5.5 μg ml −1). The concentrations of IgG to TBP complex in acute phase sera were higher and IgG to IgA1 protease was even higher, suggesting that intermediate levels of IgG to these proteins do not protect against disease.

Characterization of genetic exchanges between various highly divergent tbpBs, having occurred in Neisseria meningitidis.

Transferrin-binding protein B (TbpB) from Neisseria is an outer membrane-associated extracellular protein involved in iron capture during bacterial infection. The tbpB genes display extensive divergences throughout the open reading frame (ORF) that have presumably been selected under the pressure of the immune system. Early studies suggested that they could possibly constitute two distantly related groups of genes (sharing less than 57% identical nt). However, the analysis of one tbpB suggested the existence of a greater genetic diversity, and the occurrence of horizontal genetic exchanges leading to rearrangements of highly divergent ORFs. This study has confirmed this and revealed the occurrence of genetic exchanges having involved at least three types of very distantly related tbpBs. These rearrangements resulted from recombination events having occurred at very similar positions within an ORF region encoding a highly structured protein domain, probably due to constraints imposed by protein function and mode(s) of folding. These new data also provide valuable tools for epidemiological studies and evaluation of TbpBs as candidate vaccines.

Heterogeneity of tbpB, the transferrin-binding protein B gene, among serogroup B Neisseria meningitidis strains of the ET-5 complex.

ET-5 complex strains of Neisseria meningitidis were traced intercontinentally and have been causing hyperendemic meningitis on a worldwide scale. In an attempt to develop a fully broad cross-reactive transferrin-binding protein B (TbpB)-based vaccine, we undertook to assess the extent of variability of TbpB proteins among strains of this epidemiological complex. For this purpose, a PCR-based method was developed to study the heterogeneity of the tbpB genes from 31 serogroup B N. meningitidis strains belonging to the ET-5 complex. To define adequate primers, the tbpB gene from an ET-5 complex strain, 8680 (B:15:P1.3; isolated in Chile in 1987), was cloned and the nucleotide sequence was determined and compared to two other previously published tbpB sequences. A tbpB fragment was amplified from genomic DNA from each of the 31 strains. By this method, heterogeneity in size was observed and further characterized by restriction pattern analysis with four restriction enzymes and by sequencing tbpB genes from three other ET-5 complex strains. Four distinct tbpB gene types were identified. Fifty-five percent of the strains studied (17/31) harbored tbpB genes similar to that of strain BZ83 (B:15:-) isolated in The Netherlands in 1984. Ten of the 31 strains (32.2%) had tbpB genes close to that of strain M982. Only 3 of the 31 (9.6%) were found to harbor tbpB genes close to that of strain 8680, and finally one strain, 8710 (B:15:P1.3; isolated in Chile in 1987), was found to harbor a tbpB gene different from all the others. These results demonstrated a pronounced variability among tbpB alleles within a limited number of ET-5 complex strains collected over a 19-year period. Despite the genetic heterogeneity observed, specific antisera raised to purified Tbps from ET-5 complex strains showed broad cross-reactivity between different TbpBs both by Western blot analysis and bactericidal assay, confirming that a limited number of TbpB molecules included in a vaccine are likely to induce broadly cross-reactive antibodies against the different strains.

Characterization of the diversity and the transferrin-binding domain of gonococcal transferrin-binding protein 2.

The molecular weight heterogeneities of Tbp1 and Tbp2 among a panel of 45 gonococcal isolates were assessed. The tbpB genes from four of these strains were sequenced to characterize the Tbp2 sequence diversity among gonococci. By expressing truncated versions of gonococcal Tbp2, we delimited the extent of Tbp2 necessary for transferrin binding in a Western blot.