Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance, a Novel Antimicrobial Resistance Multilocus Typing Scheme for Tracking Global Dissemination of N. gonorrhoeae Strains.

A curated Web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeae was developed and is publicly accessible (https://ngstar.canada.ca). The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes (penA, mtrR, porB, ponA, gyrA, parC, and 23S rRNA) associated with resistance to ß-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penA sequence, combining the historical nomenclature for penA types I to XXXVIII with novel nucleotide sequence designations; the full mtrR sequence and a portion of its promoter region; portions of ponA, porB, gyrA, and parC; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) (n = 660) consisting of 29 clonal complexes (CCs) having a maximum of a single-locus variation, and 76 NG-STAR STs (n = 109) were identified as unrelated singletons. NG-STAR had a high Simpson's diversity index value of 96.5% (95% confidence interval [CI] = 0.959 to 0.969). The most common STs were NG-STAR ST-90 (n = 100; 13.0%), ST-42 and ST-91 (n = 45; 5.9%), ST-64 (n = 44; 5.72%), and ST-139 (n = 42; 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91, and ST-139 (n = 156; 92.3%); decreased susceptibility to cephalosporins was associated with NG-STAR ST-90, ST-91, and ST-97 (n = 162; 94.2%); and ciprofloxacin resistance was associated with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150, and ST-158 (n = 196; 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 (n = 106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial-resistant N. gonorrhoeae strains.

Chlamydia suis and Chlamydia trachomatis induce multifunctional CD4 T cells in pigs.

Chlamydia trachomatis infections are the most prominent bacterial sexually-transmitted disease world-wide and a lot of effort is put into the development of an effective vaccine. Pigs have been shown to be a valuable animal model for C. trachomatis vaccine development. The aim of this study was to decipher the T-cell-mediated immune response to chlamydial infections including C. trachomatis and C. suis, the chlamydia species naturally infecting pigs with a demonstrated zoonotic potential. Vaginal infection of pigs with C. suis and C. trachomatis lasted from 3 to 21days and intra-uterine infection was still present after 21days in 3 out of 5 C. suis- and 4 out of 5 C. trachomatis-inoculated animals and caused severe pathological changes. Humoral immune responses including neutralizing antibodies were found predominantly in response to C. suis starting at 14days post inoculation. The T-cell-mediated immune responses to C. trachomatis and C. suis-infections started at 7days post inoculation and consisted mainly of CD4+ T cells which were either IFN-γ single cytokine-producing or IFN-γ/TNF-α double cytokine-producing T-helper 1 cells. IL-17-producing CD4+ T cells were rare or completely absent. The T-cell-mediated immune responses were triggered by both homologous or heterologous re-stimulation indicating that cross-protection between the two chlamydia species is possible. Thus, having access to a working genital C. suis and C. trachomatis infection model, efficient monitoring of the host-pathogen interactions, and being able to accurately assess the responses to infection makes the pig an excellent animal model for vaccine development which also could bridge the gap to the clinical phase for C. trachomatis vaccine research.

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

OBJECTIVES: 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). METHODS: 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). RESULTS: Most isolates carried PBP2 mutation pattern IX (D345a, F504L, A510V, A516G and 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. CONCLUSIONS: Specific single or combined mutation patterns in penA, mtrR and porB and specific STs were associated with differences in susceptibility to ceftriaxone and cefixime.

N terminus determinants of MinC from Neisseria gonorrhoeae mediate interaction with FtsZ but do not affect interaction with MinD or homodimerization.

While bacterial cell division has been widely studied in rod-shaped bacteria, the mechanism of cell division in round (coccal) bacteria remains largely enigmatic. In the present study, interaction between the cell division inhibitor MinC from Neisseria gonorrhoeae (MinC(Ng)) and the gonococcal cell division proteins MinD(Ng) and FtsZ(Ng) are demonstrated. Protein truncation and site-directed mutagenic approaches determined which N-terminal residues were essential for cell division inhibition by MinC(Ng) using cell morphology as an indicator of protein functionality. Truncation from or mutation at the 13th amino acid of the N terminus of MinC(Ng) resulted in loss of protein function. Bioinformatic analyses predicted that point mutations of L35P and L68P would affect the alpha-helical conformation of the protein and we experimentally showed that these mutations alter the functionality of MinC(Ng). The bacterial two-hybrid system showed that interaction of MinC(Ng) with FtsZ(Ng) is abrogated upon truncation of 13 N-terminal residues while MinC(Ng)-MinD(Ng) interaction or MinC(Ng) homodimerization is unaffected. These data confirm interactions among gonococcal cell division proteins and determine the necessity of the 13th amino acid for MinC(Ng) function.

Cationic peptide of the male reproductive tract, HE2alpha, displays antimicrobial activity against Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis.

OBJECTIVES: To analyse the in vitro antimicrobial effects of synthetic HE2alpha peptide against Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis. METHODS: The HE2alpha peptide was synthesized based on the C-terminal sequence of the HE2alpha protein. The bacterial strains tested included two antibiotic-susceptible strains of N. gonorrhoeae and four antibiotic-resistant clinical isolates, as well as S. aureus ATCC 29213 and E. faecalis ATCC 29212. Susceptibility determinations were carried out either in 0.7% casamino acids for N. gonorrhoeae isolates or in 10 mM phosphate buffer for S. aureus and E. faecalis strains. Antibacterial effects were measured in a dose- and time-dependent manner. After exposure to the peptide in solution, the number of viable cells was determined by counting colony forming units (cfu). RESULTS: The HE2alpha peptide exhibited time- and dose-dependent antibacterial effects on all N. gonorrhoeae isolates tested. S. aureus and E. faecalis strains were also susceptible to the peptide. All strains tested were susceptible to the peptide at high concentrations (50 or 100 mg/L) and some strains were susceptible to a peptide concentration of 25 mg/L. CONCLUSIONS: The peptide HE2alpha, which is derived from the male urogenital tract, exhibits antibacterial activity against both gram-positive and gram-negative pathogens in vitro. The peptide is active against both antibiotic-susceptible and -resistant N. gonorrhoeae isolates. Further investigation of the antimicrobial properties of the peptide is warranted.

Conserved glycines in the C terminus of MinC proteins are implicated in their functionality as cell division inhibitors.

Alignment of 36 MinC sequences revealed four completely conserved C-terminal glycines. As MinC inhibits cytokinesis in Neisseria gonorrhoeae and Escherichia coli, the functional importance of these glycines in N. gonorrhoeae MinC (MinC(Ng)) and E. coli MinC (MinC(Ec)) was investigated through amino acid substitution by using site-directed mutagenesis. Each mutant was evaluated for its ability to arrest cell division and to interact with itself and MinD. In contrast to overexpression of wild-type MinC, overexpression of mutant proteins in E. coli did not induce filamentation, indicating that they lost functionality. Yeast two-hybrid studies showed that MinC(Ec) interacts with itself and MinD(Ec); however, no interactions involving MinC(Ng) were detected. Therefore, a recombinant MinC protein, with the N terminus of MinC(Ec) and the C terminus of MinC(Ng), was designed to test for a MinC(Ng)-MinD(Ng) interaction. Each MinC mutant interacted with either MinC or MinD but not both, indicating the specificity of glycine residues for particular protein-protein interactions. Each glycine was mapped on the C-terminal surfaces (A, B, and C) of the solved Thermotoga maritima MinC structure. We found that MinC(Ec) G161, residing in close proximity to the A surface, is involved in homodimerization, which is essential for MinC function. Glycines corresponding to MinC(Ec) G135, G154, and G171, located within or adjacent to the B-C surface junction, are critical for MinC-MinD interactions. Circular dichroism revealed no gross structural perturbations of the mutant proteins, although the contribution of glycines to protein flexibility and stability cannot be discounted. Using molecular modeling, we propose that exposed conserved MinC glycines interact with exposed residues of the alpha-7 helix of MinD.

Organization and transcription of the division cell wall (dcw) cluster in Neisseria gonorrhoeae.

A cluster of genes involved in cell division and cell wall (dcw) biosynthesis was identified in Neisseria gonorrhoeae using genomic analysis and through verification of gene order by polymerase chain reaction (PCR) analysis. The gonococcal dcw cluster consists of 17 genes, in the order 5'-mraZ-mraW-ftsI-murE-hyp1-murF- mraY-hyp2-murD-ftsW-murG-murC-ddl -ft sQ-ftsA-ftsZ-hyp3-3'. The gene organization of the dcw cluster of N. gonorrhoeae is more similar to that observed in Gram-negative rods such as Escherichia coli and Haemophilus influenzae than in Gram-positive bacteria. The cluster is characterized by several intergenic spaces. Compared with E. coli, two genes, ftsL and envA, are absent in the gonococcal dcw cluster and three hypothetical genes are novel to the cluster. The cluster is flanked by two transcriptional terminators consisting of paired neisserial uptake sequences and also includes four internal terminators, three of which are paired neisserial uptake sequences. We also found that a repeated sequence on the gonococcal genome, commonly called a Correia element, acts as the fourth transcriptional terminator. All termination sequences were shown to be fully functional by using reverse transcription PCR experiments. Transcriptional start sites upstream of ftsQ, ftsA and ftsZ were determined by primer extension and six promoters were identified; three promoters were located upstream of ftsZ in the intergenic space, two were upstream of ftsA within ftsQ and one was upstream of ftsQ within ddl. Some of these promoters were preferentially used under anaerobic conditions. The location of these promoters differed from those described in E. coli indicating dissimilar transcriptional regulation.

Stable shuttle vectors for Neisseria gonorrhoeae, Haemophilus spp. and other bacteria based on a single origin of replication.

An origin of replication (ori) was obtained from a naturally occurring beta-lactamase-producing plasmid isolated from Neisseria gonorrhoeae and used to construct shuttle vectors capable of replicating in N. gonorrhoeae, Haemophilus ducreyi, Haemophilus influenzae and Escherichia coli. Using the gonococcal proAB genes, we complemented proline-requiring N. gonorrhoeae F62 and E. coli HB101 in trans. The first demonstration of the expression of the green fluorescent protein (GFP) in either N. gonorrhoeae or H. ducreyi was shown using this vector, indicating that GFP may be a useful tool in the analysis of these organisms. This is the first report of a gonococcal vector based on a broad host range, genetically defined ori, and should facilitate the molecular analysis of gonococcal and Haemophilus genes.

Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae: expression in Escherichia coli and N. gonorrhoeae.

We cloned the cell division gene ftsZ of the gram-negative coccus Neisseria gonorrhoeae (Ng) strain CH811, characterized it genetically and phenotypically, and studied its localization in N. gonorrhoeae and Escherichia coli (Ec). The 1,179-bp ORF of ftsZ(Ng) encodes a protein with a predicted molecular mass of 41.5 kDa. Protein sequence alignments indicate that FtsZ(Ng) is similar to other FtsZ proteins and contains the conserved GTP binding motif. FtsZ homologues were identified in several N. gonorrhoeae strains and in Neisseria lactamica, Neisseria sicca, Neisseria polysaccharae and Neisseria cinerea either by Western blot or by PCR-Southern blot analysis. Attempts to inactivate the ftsZ(Ng) on the chromosome failed, indicating that it is essential for gonococcal growth. FtsZ(Ng) was synthesized in an in vitro transcription/translation system and was shown to be 43 kDa, the same size as in Western blots. Expression of the ftsZ(Ng) gene from nongonococcal promoters resulted in a filamentous phenotype in E. coli. Under controlled expression, the FtsZ(Ng)-GFP fusion protein localized at the mid-cell division site in E. coli. E. coli expressing high levels of the FtsZ(Ng)-GFP fusion protein formed filaments and exhibited different fluorescent structures including helices, spiral tubules extending from pole to pole, and regularly spaced dots or bands that did not localize at the middle of the cell. Expression of the FtsZ(Ng)-GFP fusion protein in N. gonorrhoeae resulted in abnormal cell division as shown by electron microscopy. FtsZ(Ng)-GFP fusions were also expressed in a gonococcal background using a unique shuttle vector.

A PCR assay for discriminating Neisseria gonorrhoeaebeta-lactamase-producing plasmids.

Oligonucleotide primers were developed for use in polymerase chain reaction (PCR) assays to differentiate three related, epidemic beta-lactamase-producing plasmids of Neisseria gonorrhoeae-the Asia-(7426 bp), Africa-(5599 bp) and Toronto-(5154 bp) type plasmids. One-hundred and two N. gonorrhoeae isolates with different plasmid profiles were tested-16 isolates carried the Asia plasmid, 41 isolates contained the Africa plasmid, 16 isolates contained the Toronto plasmid and 29 isolates contained no beta-lactamase-producing plasmids. Most (101/102) isolates also carried the gonococcal cryptic plasmid, while 27/102 and 44/102 isolates carried either the transfer plasmid or the tet M-containing plasmids, respectively. The assay was 100% sensitive and specific for identifying the correct plasmid type. This assay is useful for rapidly detecting the presence of gonococcal beta-lactamase-producing plasmids in clinical samples and discriminating them for epidemiological typing.

Myelin repair by Schwann cells in the regenerating goldfish visual pathway: regional patterns revealed by X-irradiation.

In the regenerating goldfish optic nerves, Schwann cells of unknown origin reliably infiltrate the lesion site forming a band of peripheral-type myelinating tissue by 1-2 months, sharply demarcated from the adjacent new CNS myelin. To investigate this effect, we have interfered with cell proliferation by locally X-irradiating the fish visual pathway 24h after the lesion. As assayed by immunohistochemistry and EM, irradiation retards until 6 months formation of new myelin by Schwann cells at the lesion site, and virtually abolishes oligodendrocyte myelination distally, but has little or no effect on nerve fibre regrowth. Optic nerve astrocyte processes normally fail to re-infiltrate the lesion, but re-occupy it after irradiation, suggesting that they are normally excluded by early cell proliferation at this site. Moreover, scattered myelinating Schwann cells also appear in the oligodendrocyte-depleted distal optic nerve after irradiation, although only as far as the optic tract. Optic nerve reticular astrocytes differ in various ways from radial glia elsewhere in the fish CNS, and our observations suggest that they may be more permissive to Schwann cell invasion of CNS tissue.

Detection of the tetM determinant in Neisseria gonorrhoeae using a non-radioactively labelled oligonucleotide probe.

Three oligonucleotide probes, complementary to tetM sequences, were labelled non-radiometrically using the DIG-oligonucleotide tailing kit and evaluated for their specificity for the detection of plasmid mediated tetracycline resistance in Neisseria gonorrhoeae. Only Probe 3, 5'-GCT CAA CAA TTC TGT TCC AGC-3', was specific for tetM. It hybridized with the tetM-containing 25.2-MDa plasmids from all of the 232 TRNG and the 130 PP/TRNG isolates used in the study. Its sensitivity, determined by dot-blot hybridization, was 0.1 pg of pJ13 plasmid DNA or 10(4) cells. It did not hybridize with the DNA from non-PPNG, CMRNG and tetracycline susceptible isolates from seven other Neisseria species (N. meningitidis, N. subflava, N. cinerea, N. lactamica, N. sicca, N. mucosa, and N. flavescens), Moraxella spp. and Haemophilus influenzae. Probe 3 also hybridized to DNA of three tetracycline resistant P. magnus (MIC = 16 micrograms ml-1) isolates which presumptively carried the tetM determinant. Therefore, probe 3 can be used by reference laboratories as a confirmatory test for TRNG, as well as isolates from other genera containing the tetM determinant.

Typing by serovar, antibiogram, plasmid content, riboprobing, and isoenzyme typing to determine whether Neisseria gonorrhoeae isolates requiring proline, citrulline, and uracil for growth are clonal.

Neisseria gonorrhoeae isolates requiring proline, citrulline, and uracil for growth (PCU-) have homogeneous phenotypes; most are plasmid-free, belong to few serovars, and are significantly associated with intermediate levels of susceptibility to penicillin, tetracycline, erythromycin, and cefoxitin. Because of their lack of variation by these criteria, molecular typing methods, ribotyping (restriction fragment length polymorphism [RFLP] of rRNA genes), and multilocus enzyme electrophoresis were explored as tools for further distinguishing PCU- isolates. By ribotyping, selected PCU- isolates could be separated into four groups on the basis of the hybridization patterns (RFLPs) of SmaI- and AvaII-digested DNA with probes containing rRNA sequences. Most of the isolates (18 of 23 isolates) belonged to a single RFLP (group I). One isolate each was in groups II and IV, and three isolates were in group III. All isolates except one, isolate NS791, had similar multilocus enzyme electrophoresis patterns. Strain NS791 was unusual in that it contained a variant cryptic plasmid with an insert in the 0.46-kb MspI-HinfI fragment of the 4.2-kb plasmid, it was the only isolate belonging to RFLP group IV, and it differed in its multilocus enzyme electrophoresis pattern, having different mobilities for glyceraldehyde phosphate dehydrogenase, phosphoglucose isomerase, 6-phosphogluconate dehydrogenase, and glutamate dehydrogenase. Serovars of PCU- isolates appeared to be more indicative of strain divergence than RFLP or isoenzyme typing. Multilocus enzyme electrophoresis indicated that PCU- isolates are clonal.

Evaluation of a urease-based confirmatory enzyme-linked immunosorbent assay for diagnosis of Neisseria gonorrhoeae.

A new urease-based enzyme-linked immunosorbent assay utilizing novel monoclonal antibodies was evaluated for the culture confirmation of Neisseria gonorrhoeae, with 270 isolates of N. gonorrhoeae, 56 isolates of diverse Neisseria spp., and 29 Moraxella isolates. The test was highly specific (100.00%) and sensitive (97.83%). No cross-reactions were observed with any of the Neisseria or Moraxella isolates tested. Fifty percent (3 of 6) of the false-negative results were obtained with isolates of serovar IA-4, a serovar rarely encountered in North America.

Myelination of regenerated axons in goldfish optic nerve by Schwann cells.

This study uses immunohistochemistry and EM to examine the site of injury in goldfish optic nerve during axonal regeneration. Within seven days of nerve crush axons begin to regrow and a network of GFAP+ reactive astrocytes appears in the nerve on either side of the injury. However, the damaged area remains GFAP-. By 42 days after nerve crush, the sheaths of new axons acquire myelin marker 6D2, and the crush area becomes populated by a mass of longitudinally-orientated S-100+ cells. Ultrastructurally, the predominant cells in the crush area bear a strong resemblance to peripheral nerve Schwann cells; they display a one-to-one association with myelinated axons, have a basal lamina and are surrounded by collagen fibres. It is proposed that these cells are Schwann cells which enter the optic nerve as a result of crush, where they become confined to the astrocyte-free crush area.

A polyclonal antibody to goldfish neuronal 145 kDa intermediate filament protein.

A polyclonal antibody raised to a protein from goldfish optic tectum recognises, immunohistochemically, axons throughout normal goldfish visual pathway. In goldfish with injured optic nerve, this antibody recognises degenerating neuronal debris as well as regenerating fibres. On immunoblot, the antibody recognises, primarily, a neuronal intermediate filament protein in the region of 145 kDa. Such an antibody should prove useful in studies pertaining to goldfish visual pathway.