Gonococcal Strains Research Articles

Most but not all laboratories can detect the recently emerged Neisseria gonorrhoeae porA mutants – results from the QCMD 2013 N. gonorrhoeae external quality assessment programme

We describe the results of the Quality Control for Molecular Diagnostics 2013 Neisseria gonorrhoeae external quality assessment programme that included an N. gonorrhoeae strain harbouring an N. meningitidis porA gene which causes false-negative results in molecular diagnostic assays targeting the gonococcal porA pseudogene. Enhanced awareness of the international transmission of such gonococcal strains is needed to avoid false-negative results in both in-house and commercial molecular diagnostic assays used in laboratories worldwide, but particularly in Europe.

Induction of Endotoxin Tolerance by Pathogenic Neisseria Is Correlated with the Inflammatory Potential of Lipooligosaccharides and Regulated by MicroRNA-146a

In this article, we report that retreatment of human monocytic THP-1 cells and primary monocytes with pathogenic Neisseria or with purified lipooligosaccharides (LOS) after previous exposure to LOS induced immune tolerance, as evidenced by reduced TNF-α and IL-1β cytokine expression. LOS that we have previously shown to vary in their potential to activate TLR4 signaling, which was correlated with differences in levels of lipid A phosphorylation, had similarly variable ability to induce tolerance. Efficacy for induction of tolerance was proportional to the level of lipid A phosphorylation, as LOS from meningococcal strain 89I with the highest degree of phosphorylation was the most tolerogenic following retreatment with LOS or whole bacteria, compared with LOS from gonococcal strains 1291 and GC56 with reduced levels of phosphorylation. Hydrogen fluoride treatment of 89I LOS to remove phosphates rendered the LOS nontolerogenic. Tolerance induced by the more highly inflammatory meningococcal LOS was correlated with significantly greater downregulation of p38 activation, greater induction of the expression of A20 and of microRNA-146a, and greater reductions in IL-1R-associated kinase 1 and TRAF6 levels following LOS retreatment of cells. The role of miR-146a in regulation of induction of TNF-α was confirmed by transfecting cells with an inhibitor and a mimic of miR-146a. Our results provide a mechanistic framework for understanding the variable pathophysiology of meningococcal and gonococcal infections given that after an initial exposure, greater upregulation of microRNA-146a by more highly inflammatory LOS conversely leads to the suppression of immune responses, which would be expected to facilitate bacterial survival and dissemination.

Unique combined penA/mtrR/porB mutations and NG-MAST strain types associated with ceftriaxone and cefixime MIC increases in a ‘susceptible’ Neisseria gonorrhoeae population

To determine which mutations in penA, mtrR and porB are implicated in increasing minimum MICs of ceftriaxone and cefixime in a susceptible gonococcal population and to ascertain associations with gonococcal strain types (STs). One hundred and forty-six Neisseria gonorrhoeae isolates formed two extended-spectrum cephalosporin susceptibility groups: group 1 isolates with cefixime and ceftriaxone MICs of 0.0005-0.016 mg/L; and group 2 isolates with cefixime MICs of 0.03-0.125 mg/L (n = 24) and ceftriaxone MICs of 0.03-0.06 mg/L (n = 23). Mutation patterns in penicillin-binding protein 2 (PBP2; penA), multiple transfer resistance repressor (MtrR; mtrR) and porin B (PorB; porB) were ascertained by DNA sequence and bioinformatic analysis. STs were determined using N. gonorrhoeae multiantigen sequence typing (NG-MAST). Most isolates carried PBP2 mutation pattern IX (D345a, F504L, A510V, A516Gand P551L; 50/146, 34.2%), a G45D substitution in MtrR (37.7%) and a wild-type (WT) sequence for PorB (43.2%). Group 2 gonococcal isolates were significantly associated with: penA pattern IX; dual mutations in the promoter (A-) and DNA dimerization domain (H105Y) of MtrR; and G120K;A121D substitutions in PorB. There were 50 combined penA/mtrR/porB mutation patterns, with corresponding patterns I/WT/WT and IX/G45D/G120K;A121D predominating. Gonococci susceptible to ceftriaxone and cefixime were significantly associated with NG-MAST ST 25 (33/36; 92%) and the combined penA/mtrR/porB mutation pattern I/WT/WT. No combined mutation pattern or specific ST was associated with elevated ceftriaxone MICs. NG-MAST ST 3654 was significantly associated with the pattern IX/G45D/G120K;A121D and cefixime group 2 isolates. Specific single or combined mutation patterns in penA, mtrR and porB and specific STs were associated with differences in susceptibility to ceftriaxone and cefixime.

Control of pili and sialyltransferase expression in Neisseria gonorrhoeae is mediated by the transcriptional regulator CrgA

Contact-regulated gene A (CrgA) is a transcriptional regulator present in the pathogenic Neisseria that functions as both an activator and a repressor of transcription following contact with host cells. While its mechanism of action has been studied extensively in Neisseria meningitidis, the specific subset of genes that CrgA targets has been debated. Although the majority of these constitute virulence genes, suggesting that CrgA is important in pathogenesis, no study to date has examined the effects of CrgA in Neisseria gonorrhoeae. In this report, we generated a knockout mutant of crgA (ΔcrgA) in the serum-sensitive gonococcal strain F62. crgA deletion resulted in a reduction in the transcript and protein levels of the primary pilin component pilE via mechanisms that were both contact-dependent and -independent. In contrast, ΔcrgA overexpressed the main determinant of serum resistance in F62, lipooligosaccharide sialyltransferase (Lst). CrgA-mediated lst repression was direct as both recombinant and native CrgA bound to the lst promoter at multiple locations in EMSA and ChIP assays respectively. The increase in Lst levels associated with crgA deletion correlated with enhanced protection against killing by normal human serum. These data suggest a role for CrgA in virulence regulation during both cell adherence and planktonic growth.

Severe Conjunctivitis Due to Multidrug‐ResistantNeisseria gonorrhoeaeand Adenovirus 53 Coinfection in a Traveler Returning From Thailand

A male traveler returning from Thailand with severe bilateral conjunctivitis was tested for causative pathogens by culture and polymerase chain reaction in late 2010. The culturally grown Neisseria gonorrhoeae strain was resistant against penicillin, ciprofloxacin, and tetracycline. The patient was also found to have an eye infection with the unusual and likely recombinant adenovirus type 53. Besides multidrug-resistant gonococcal strains the unusual adenovirus strain is found circulating in Asia and both pathogens may be a risk for travelers.

Construction of a recombinant Neisseria gonorrhoeae strain with rmp gene deletion and study on the bactericidal activities of its antibodies

Objective To study the role of the outer membrane protein Rmp of Neisseria gonor- rhoeae strain in immunosuppression and the strategy of eliminating it. Methods The rmp gene of Neisseria gonorrhoeae strain was amplified by PCR and inserted into pMD19-T vector. The recombinant vector pMD19 Armp ：： Kan containing Kan and the 5＇-and 3＇-flanking regions of rmp （ Armp ：： Kan） was constructed by replacing 200 nucleotide residues of pMD19-rmp with kanamycin resistance gene Kan and transformed into Neisseria gonorrhoeae WHO-A strain. PCR and Western blot assay were used to screen and identify the re- combinant mutant strains that could not express Rmp. Mice were immunized with mutant strains and bacteri- cidal activities of the immune sera were detected by antibody-mediated complement-dependent cytotoxicity assay. Results The mutant strains that could not encode Rmp were successfully constructed. Antibodies in- duced by mutant strains showed stronger bactericidal activity against Neisseria gonorrhoeae in comparison with those induced by wild strains. Conclusion The recombinant Neisseria gonorrhoeae strain with rmp gene de- letion might eliminate the immunosuppressive effects of Rmp expressed in wild gonococcal strains, which provides a reference for further development of novel live attenuated whole-cell vaccines of Neisseria gonor- rhoeo, e. Key words: Neisseria gonorrhoeae; Homologous recombination; rmp gene deletion mutants; Live attenuated vaccine

Neisseria Prophage Repressor Implicated in Gonococcal Pathogenesis

Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, can infect and colonize multiple mucosal sites in both men and women. The ability to cope with different environmental conditions requires tight regulation of gene expression. In this study, we identified and characterized a gonococcal transcriptional regulatory protein (Neisseria phage repressor [Npr]) that was previously annotated as a putative gonococcal phage repressor protein. Npr was found to repress transcription of NGNG_00460 to NGNG_00463 (NGNG_00460-00463), an operon present within the phage locus NgoΦ4. Npr binding sites within the NGNG_00460-00463 promoter region were found to overlap the -10 and -35 promoter motifs. A gonococcal npr mutant demonstrated increased adherence to and invasion of human endocervical epithelial cells compared to a wild-type gonococcal strain. Likewise, the gonococcal npr mutant exhibited enhanced colonization in a gonococcal mouse model of mucosal infection. Analysis of the gonococcal npr mutant using RNA sequence (RNA-seq) analysis demonstrated that the Npr regulon is limited to the operon present within the phage locus. Collectively, our studies have defined a new gonococcal phage repressor protein that controls the transcription of genes implicated in gonococcal pathogenesis.

P3.263 Do Men Harbour the Same Strain of Gonococcus at Multiple Anatomical Sites?

BackgroundAll N. gonorrhoeae clinical isolates in Scotland are routinely typed by Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). The frequency with which individuals harboured the same N. gonorrhoeae strain at >...

PL01.1 Antimicrobial Resistance in Neisseria Gonorrhoeae and Possible Emergence of Untreatable Gonorrhoea: How, When and Essential Actions?

The WHO estimated 106 million gonorrhoea cases in 2008 globally and antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major public health concern that compromises effective treatment and disease control...

P2.096 Evaluation of Gonococcal Resistance in Austria

BackgroundThe Austrian Society for Sexually Transmitted Diseases started a surveillance project in 2010 to evaluate the gonococcal resistance in Austria.MethodsIn 2010 and 2011 a national network of 39 centres was...

P5.102 Antimicrobial Susceptibility of Neisseria Gonorrhoeae in Nanjing, China, 2011–2012

BackgroundA study of gonococcal transmission from men to their female contacts has been under investigation at the STD clinic in Nanjing, China since April 2011. Men with symptomatic urethral discharge...

P5.101 Rising Trend of Antimicrobial Resistance Among Neisseria Gonorrhoeae Isolates in a Central Delhi Tertiary Care Hospital

BackgroundGonorrhoea continues to be a common sexually transmitted disease (STD) in developing countries. Over the last decade, N.gonorrhoeae has developed resistance against antimicrobial agents such as penicillin, tetracycline and quinolones...

P3.120 Risk Factors, Antimicrobial Susceptibility Profiles and Public Health Implications of Patients Presenting with Pharyngeal Gonococcal Infection in England and Wales

BackgroundPharyngeal infection with Neisseria gonorrhoeae is usually asymptomatic and often under-diagnosed. It could therefore be an important source of ongoing transmission particularly among men who have sex with men (MSM)....

Épidémiologie actuelle des infections sexuellement transmissibles bactériennes en France

Sexually transmitted infections (STIs) remains a major problem of public health in France. Voluntary networks of physicians (RésIST) and laboratories (Rénago, Rénachla, lymphogranuloma venereum: LGV network) produce indicators showing the evolution of the main bacterial STIs. In 2010, the main findings were the following. The number of gonococcal infections has increased throughout the decade 2000 to 2010. The decrease in susceptibility of gonococcal strains to first-line antibiotics (extended-spectrum cephalosporins) needs to keep great attention. The number of screening and diagnosis of chlamydial urogenital infections also continues to rise in both sexes, particularly due to increased screening among young people. The relatively stable number of cases of early syphilis and of rectal LGV needs to be confirmed over the coming years. Both of these STIs affect overwhelmingly homo/bisexual men. There is still a high level of HIV co-infection with LGV and syphilis, and to a lesser extent with gonorrhea. We observe that condom use is still inadequate, especially during oral sex.

Mating Pair Formation Homologue TraG Is a Variable Membrane Protein Essential for Contact-Independent Type IV Secretion of Chromosomal DNA by Neisseria gonorrhoeae

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the surrounding milieu. The DNA is effective in transforming gonococci in the population, and this mechanism of DNA donation may contribute to the high degree of genetic diversity in this species. Similar to other F-like T4SSs, the gonococcal T4SS requires a putative membrane protein, TraG, for DNA transfer. In F-plasmid and related systems, the homologous protein acts in pilus production, mating pair stabilization, and entry exclusion. We characterized the localization, membrane topology, and variation of TraG in N. gonorrhoeae. TraG was found to be an inner-membrane protein with one large periplasmic region and one large cytoplasmic region. Each gonococcal strain carried one of three different alleles of traG. Strains that carried the smallest allele of traG were found to lack the peptidoglycanase gene atlA but carried a peptidoglycan endopeptidase gene in place of atlA. The purified endopeptidase degraded gonococcal peptidoglycan in vitro, cutting the peptide cross-links. Although the other two traG alleles functioned for DNA secretion in strain MS11, the smallest traG did not support DNA secretion. Despite the requirement for a mating pair stabilization homologue, static coculture transformation experiments demonstrated that DNA transfer was nuclease sensitive and required active uptake by the recipient, thus demonstrating that transfer occurred by transformation and not conjugation. Together, these results demonstrate the TraG acts in a process of DNA export not specific to conjugation and that different forms of TraG affect what substrates can be transported.

Transcription of a cis-acting, Noncoding, Small RNA Is Required for Pilin Antigenic Variation in Neisseria gonorrhoeae

The strict human pathogen Neisseria gonorrhoeae can utilize homologous recombination to generate antigenic variability in targets of immune surveillance. To evade the host immune response, N. gonorrhoeae promotes high frequency gene conversion events between many silent pilin copies and the expressed pilin locus (pilE), resulting in the production of variant pilin proteins. Previously, we identified a guanine quartet (G4) structure localized near pilE that is required for the homologous recombination reactions leading to pilin antigenic variation (Av). In this work, we demonstrate that inactivating the promoter of a small non-coding RNA (sRNA) that initiates within the G4 forming sequence blocks pilin Av. The sRNA promoter is conserved in all sequenced gonococcal strains, and mutations in the predicted transcript downstream of the G4 forming sequence do not alter pilin Av. A mutation that produces a stronger promoter or substitution of the pilE G4-associated sRNA promoter with a phage promoter (when the phage polymerase was expressed) produced wild-type levels of pilin Av. Altering the direction and orientation of the pilE G4-associated sRNA disrupted pilin Av. In addition, expression of the sRNA at a distal site on the gonococcal chromosome in the context of a promoter mutant did not support pilin Av. We conclude that the DNA containing the G-rich sequence can only form the G4 structure during transcription of this sRNA, thus providing a unique molecular step for the initiation of programmed recombination events.

Phosphoethanolamine Residues on the Lipid A Moiety of Neisseria gonorrhoeae Lipooligosaccharide Modulate Binding of Complement Inhibitors and Resistance to Complement Killing

Loss of phosphoethanolamine (PEA) from the lipid A of gonococcal strain FA19 results in increased sensitivity to killing by the classical pathway of complement. Here we demonstrate that loss of PEA from lipid A diminishes binding of the complement regulatory protein C4b binding protein (C4BP) to the FA19 porin B (PorB), providing a molecular basis to explain the susceptibility of an lptA null strain of FA19 to killing by normal human serum (NHS). Loss of PEA from lipid A in three additional gonococcal strains that expressed diverse PorB molecules also resulted in decreased C4BP binding, increased deposition of C4b, and increased susceptibility to killing by NHS. Complementation of lptA null strains with lptA restored C4BP binding, decreased C4b deposition, and increased resistance to killing by NHS. These effects of lipid A PEA on C4BP binding to gonococcal PorB and serum resistance were simulated when gonococcal PorB was expressed in a meningococcal background. Loss of PEA from lipid A also affected binding of the alternative pathway regulator factor H (fH) to PorB of some strains. For instance, PorB molecules of lptA null mutants of strains 252 and 1291 bound less fH than those of their parent strains when lipooligosaccharide (LOS) was sialylated, whereas PorB molecules of lptA null mutants of strains FA1090 and 273 retained the ability to bind fH when LOS was sialylated. These data indicate that replacement of lipid A with PEA alters binding of C4BP and fH to PorB and contributes to the ability of gonococci to resist complement-mediated killing.

Sequence Features Contributing to Chromosomal Rearrangements in Neisseria gonorrhoeae

Through whole genome sequence alignments, breakpoints in chromosomal synteny can be identified and the sequence features associated with these determined. Alignments of the genome sequences of Neisseria gonorrhoeae strain FA1090, N. gonorrhoeae strain NCCP11945, and N. gonorrhoeae strain TCDC-NG08107 reveal chromosomal rearrangements that have occurred. Based on these alignments and dot plot pair-wise comparisons, the overall chromosomal arrangement of strain NCCP11945 and TCDC-NG08107 are very similar, with no large inversions or translocations. The insertion of the Gonococcal Genetic Island in strain NCCP11945 is the most prominent distinguishing feature differentiating these strains. When strain NCCP11945 is compared to strain FA1090, however, 14 breakpoints in chromosomal synteny are identified between these gonococcal strains. The majority of these, 11 of 14, are associated with a prophage, IS elements, or IS-like repeat enclosed elements which appear to have played a role in the rearrangements observed. Additional rearrangements of small regions of the genome are associated with pilin genes. Evidence presented here suggests that the rearrangements of blocks of sequence are mediated by activation of prophage and associated IS elements and reintegration elsewhere in the genome or by homologous recombination between IS-like elements that have generated inversions.

Longitudinal analysis of the evolution and dissemination of Neisseria gonorrhoeae strains (Saskatchewan, Canada, 2005 to 2008) reveals three major circulating strains and convergent evolution of ciprofloxacin and azithromycin resistance.

A longitudinal study combining multilocus sequence typing with molecular evolutionary analysis determined the distribution, population structure, and evolution of antibiotic resistance in Neisseria gonorrhoeae isolates in Saskatchewan that were collected between 2005 and 2008. Of 195 gonococcal isolates examined, 29 sequence types (STs) were identified with 3 major circulating strains (ST-1 through ST-3) comprising 52% of all gonococcal isolates studied. The prevalences, persistence, distribution patterns, and clonalities of these isolates strongly suggest that gonorrhea endemicity within this broad geographic region was driven by these 3 circulating strains. ST-1 exhibited a significantly (P = 0.001) higher prevalence throughout the study than did the others, accounting for ∼25% of the tested isolates each year. The spatial distributions of the gonococcal strains indicated that ST-1 in 2007 entered a linear component of the sexual network, reaching the remote north and resulting in the further spread and maintenance of infection. Ciprofloxacin and azithromycin resistances were observed in distantly related gonococcal lineages, clearly indicating the convergent acquisition of these antibiotic-resistant phenotypes. In addition, all ciprofloxacin- and azithromycin-resistant lineages were found at the edges of the minimum spanning tree, far from the major lineages, suggesting that these antibiotic phenotypes were most likely introduced into the province. In contrast, resistance to penicillin was found mostly in the endemic gonococcal lineages, suggesting that penicillin resistance was probably acquired in Saskatchewan as a result of spontaneous mutations in already-established lineages. Tetracycline resistance was present in all STs except one, indicating its ubiquitous nature in the gonococcal population studied.

Novel meningococcal 4CMenB vaccine antigens – prevalence and polymorphisms of the encoding genes in Neisseria gonorrhoeae

The first cross-protective Neisseria meningitidis vaccine (focus on serogroup B), the protein-based 4 component meningococcus serogroup B (4CMenB), includes the New Zealand outer membrane vesicle and three main genome-derived neisserial antigens (GNAs). These GNAs are fHbp (fused to GNA2091), NHBA (fused to GNA1030) and NadA. In this study, the prevalence and polymorphisms of the nucleotide and amino acid sequences of the 4CMenB antigens in a temporally and geographically diverse collection of N. gonorrhoeae isolates (n = 111) were investigated. All the examined GNA genes, except the nadA gene, were present in all gonococcal isolates. However, 25 isolates contained premature stop codons in the fHbp gene and/or the nhba gene, resulting in truncated proteins. Compared with the 4CMenB antigen sequences in reference strain MC58, the gonococcal strains displayed 67.0-95.4% and 60.9-94.9% identity in nucleotide sequence and amino acid sequence, respectively, in the equivalent GNA antigens. The absence of NadA, lack of universal expression of fHbp and NHBA and the uncertainty regarding the surface exposure of fHbp as well as the function of NHBA in N. gonorrhoeae will likely limit the use of the identical 4CMenB antigens in a gonococcal vaccine. However, possible cross-immunity of 4CMenB with gonococci and expression and function of the equivalent gonococcal GNAs, as well as of more appropriate GNAs for a gonococcal vaccine, need to be further examined.