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.

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.

Spectral Sensitivity of the Scallop Pecten maximus.

The spectral sensitivity of the scallop Pecten maximus was determined behavorially; the shadow reflex was used as the index of sensitivity. The photopic visibility curve displays two prominent peaks, one at approximately 480 mmicro, and the other with wavelength maximum at approximately 540 mmicro. Reduction of background illumination results in a great increase in sensitivity at 480 mmicro relative to the 540-mmicro peak. This suggests that the scallop may possess photoreceptors that behave like rods in vertebrates. sensitivity of this animal.

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.

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.

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.

Postnatal developmental profiles of filamentous actin and of 200 kDa neurofilament polypeptide in the visual cortex of light- and dark-reared rats and their relationship to critical period plasticity.

The concept of critical period plasticity in rat visual cortex has been studied in terms of changes in the level of filamentous actin and of the 200 kDa neurofilament polypeptide. Our results suggest that the postnatal developmental profile of filamentous actin is affected by visual experience, as a consequence of eye-opening. No such correlation, however, is detected for the 200 kDa neurofilament polypeptide. The significance of these findings in relationship to neuronal plasticity is discussed in terms of changes in the state and equilibrium conditions of the cytoskeletal proteins.

Strategies for molecular characterisation of methicillin- and gentamicin-resistant Staphylococcus aureus in a Canadian nosocomial outbreak.

Sixteen methicillin-resistant Staphylococcus aureus (MRSA) isolates, from a single nosocomial outbreak, were tested for molecular and phenotypic relationships. Two of the 16 outbreak strains were gentamicin resistant (Gmr) and the plasmids that they carried were characterised by reverse field electrophoresis, restriction endonuclease analysis and gene hybridisation. The gentamicin-resistant (Gmr) strains harboured two plasmids, a Gmr plasmid of 36.5 kb and a cryptic plasmid of 25.4 kb, whereas the other 14 isolates contained only the cryptic plasmid. Gentamicin resistance was encoded by a 2.5-kb HindIII fragment of the 32.8-kb plasmid and is similar to the 2.5-kb HindIII fragment also described for S. aureus Gmr plasmids from Australia and the USA. The Gmr plasmid was non-conjugal and was cured by ethidium bromide at a frequency of 4%. Two MRSA strains isolated subsequently from the same hospital were also Gmr and had identical plasmid and restriction endonuclease profiles to the two Gmr strains studied initially. Two other S. aureus isolates from the original carrier detected in this study and from his son were methicillin and gentamicin susceptible and had novel profiles. Since large plasmids show anomalous migration in agarose gels, more definitive analyses than simple plasmid identification should be considered when studying nosocomial outbreaks.

Goldfish retina and tectum influence each other's growth activity during regrowth of the retinotectal projection.

Variations in retinal and tectal growth activity, during regrowth of the goldfish retinotectal projection, were monitored by measuring the rates of incorporation of [14C]leucine into soluble protein and tubulin-enriched fractions at different times after crushing the optic nerves. Other experiments tested for growth-modulating interactions between tectum and retina. Here we studied how the absence of one of these structures (i.e. tectal ablation or eye removal) affected the profile of biosynthetic activity in the other. Experiments were also conducted on groups of fish in which the tectum was reinnervated by a half-retina (either half-nasal (1/2 N) or half-temporal (1/2 T) retina). This was done to ascertain if growth interactions between retina and tectum display any position-dependent differences that may be relevant to retinotopic ordering during regeneration. Our studies have revealed that: the retina and tectum of 1/2 T and 1/2 N groups differ in their growth responses during regeneration of the visual pathway: the tectum may exert a stimulatory and at other times an inhibitory influence on retinal protein synthesis; and retina and tectum display a bimodal profile of biosynthetic activity during regeneration that coincides with two stages of increased cell division (primarily glia) which other workers have found occurs in the tectum and tract during regeneration of the retinotectal projection. Indeed it seems there may be a link between this glial proliferation and the neurotrophic and guiding influences which tectum and retina exert upon one another during regeneration.

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.

Preferential histochemical staining of protoplasmic and fibrous astrocytes in rat CNS with GFAP antibodies using different fixatives.

Serial sections of rat brain and spinal cord were fixed in either acid-alcohol or 4% paraformaldehyde, and stained for visualization of astrocytes using GFAP antibodies. With paraformaldehyde, GFAP-positive astrocytes were visualised almost exclusively in the grey matter of all above tissues. In sharp contrast, acid-alcohol treatment gave intensely stained GFAP-containing astrocytes in the white matter. Since fibrous astrocytes are mainly located in the white matter and protoplasmic astrocytes are located in the grey matter, it is concluded that acid-alcohol is a good fixative for fibrous astrocytes while paraformaldehyde is a better fixative for protoplasmic astrocytes.

Anti-goldfish glial fibrillary acidic protein (GFAP) recognises astrocytes from rat CNS.

A polyclonal antibody to goldfish GFAP recognises, immunohistochemically, astrocyte populations in rat brain, spinal cord and optic nerve. The pattern of staining compares favourably with that obtained using a polyclonal anti-human GFAP or a monoclonal anti-porcine GFAP. These results are consistent with the notion that GFAP is well conserved in vertebrate phylogeny.

Identification of astrocyte- and oligodendrocyte-like cells of goldfish optic nerves in culture.

Fish glial cells were obtained from cultivated segments of the optic nerve and raised in vitro. Two types of cells were identified as astrocyte- and oligodendrocyte-like glia by the monoclonal antibody Mab O1 (specific for oligodendrocytes) and the rabbit serum anti-goldfish glial fibrillary acidic protein (anti-G-GFAP). Cells of compact morphology were rare, and anti-G-GFAP positive and O1 negative. Multipolar cells in 5-day-old cultures were anti-G-GFAP but rarely O1 positive. In 5-week-old cultures, however, roughly 75% of the multipolar cells were double-labeled with both anti-G-GFAP and O1; 10% were single labeled with Mab O1 and 15% with anti-G-GFAP, respectively.

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.

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.