Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae.

BACKGROUND: The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. OBJECTIVES: To characterize the genetic pathways leading to high-level azithromycin resistance. METHODS: A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. RESULTS: Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. CONCLUSIONS: This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

[Secondary benefits of cognitive rehabilitation for a chronic ethylic patient: effects on disorder consciousness, motivation, and global therapeutic cooperation]. / Bénéfices secondaires d'une réhabilitation cognitive réalisée chez un patient souffrant d'un éthylisme chronique: effets sur la conscience des troubles, la motivation, et la coopération globale aux soins.

INTRODUCTION: Alcoholism causes psychological, behavioral and cognitive symptoms that need to be addressed together. The neuropsychological alterations among alcohol-dependent people are considered to make the therapeutic work complex and longer. A cognitive rehabilitation program is sometimes difficult to achieve with these patients. Functional results are often difficult to anticipate. However, the consequences of this therapeutic approach are multiple and there are many interactions between psycho-affective, behavioral and cognitive components. A neuropsychological approach can be used like a tool to improve metacognition. A bad contribution to treatment programs is often secondary to the illusion of a satisfying intellectual functioning. Patients' motivation for the therapeutic work is very changeable. A complete consciousness of impairments can help them to stay involved. CASE REPORT: The following case shows the cognitive effects and secondary benefits associated with a neuropsychological work, which was carried out by a chronic ethylic patient with severe physical and cognitive symptoms. The patient aged 50, with a good qualification level (scientific section in the final year of secondary school, with no diploma, then attended a training program to become a croupier) was suffering from chronic alcoholism since his adolescence. He arrived in the closed unit after many hospitalizations in psychiatric and hepato-gastroenterology units. He had been showing mental confusion. He presented a frontal and subcortical profile of alcohol-related dementia according to Oslin's criteria. MRI revealed global cerebral atrophy, more pronounced on the fronto-parietal cortex with cerebellar leukoencephalopathy, but no pontine central myelinolysis. The neurocognitive program had two main lines: reducing attentional, executive and graphical deficits with training exercises (individual and group sessions) and compensating memory, and executive disorders with an external aid. The cognitive program had been assessed by means of repeated psychometric measures, behavior and metacognition estimated by direct clinical observations. The cognitive remediation was carried out during a 10-month hospitalization, and then in an outpatient rehabilitation setting over a 12-month period. The external individual sessions were associated with medical consultations and support for reintegration at home provided by the mobile psychiatric team. RESULTS: The test results showed a significant improvement in attentional processes and executive functions. On graphic level, his writing was recovered after 10 months. Impairment within episodic memory processes-encoding was observed, and prospective memory was reinforced by external aids. At the end of the program, the use of an agenda proved to be effective even if updating was difficult once back home. Although a part of these effects could be expected, their psychological influence on the patient must be underlined: his perception of the alcohol related problem had been modified with more consciousness of the neurobiological consequences and a strong desire of personal implication. He worked a lot on his own (always under supervision) on cognitive exercises and succeeded in remaining abstinent for nearly 2 years. He died of complications of acute hepatitis. DISCUSSION: All the cognitive assessments and rehabilitation results seem to have increased his participation in the global therapeutic care. Therapeutic tools used for cognitive work give a concrete picture of the consequences of alcohol consumption and the necessary time to retrieve brain capacities. Visible improvements in terms of reduction in disability-handicaps encouraged the patient, valued his efforts, and increased his determination to solve his alcohol related problem. Thus, cognitive programs can help to reduce passiveness and develop activeness. This patient regarded therapeutic difficulties as a challenge, not as an obstacle. We can reasonably assume the effect of the cognitive rehabilitation on the Persistence of the Cloninger's biosocial model. This increase in temperament corresponds to a certain form of tenacity, which would facilitate abstinence.

Chlamydial infection: an accurate model for opportunistic screening in general practice.

OBJECTIVES: To estimate the prevalence of Chlamydia trachomatis in women in general practice and to assess risk factors associated with infection. METHODS: The study was carried out in 2001-2 in different general practices in Antwerp, Belgium. Sexually active women, visiting their general practitioner for routine gynaecological care (mostly pill prescription or PAP smear), were offered opportunistic screening for chlamydia. 787 participants aged 15-40 delivered a self taken vaginal sample and filled in a questionnaire which included questions on demographic variables, urogenital symptoms, sexual history, and sexual behaviour. Samples were tested for presence of chlamydial DNA by means of a ligase chain reaction (LCR) assay, and positives were confirmed by two other amplification assays (PCR and SDA). RESULTS: Overall prevalence was 5.0% (95% CI: 3.5 to 6.5). Determinants of infection in logistic regression analysis were age 18-27 years, >1 partner in the past year, no use of contraceptives, frequent postcoital bleeding, having a symptomatic partner, painful micturition, and living in the inner city. The area under the ROC curve in the full model was 0.88. Selective screening based on a combination of the five first determinants detects 92.3% of infections in this sample; 37.5% of the population would need to be screened. CONCLUSION: Targeted screening for chlamydial infection is possible, even in a heterogeneous group of general practice attendants. Implementing this model would require considerable communication skills from healthcare providers.

Interpreting the rising incidence of meningococcal disease in Belgium: the contribution of molecular typing.

During a period of increasing meningococcal disease incidence in Belgium, all 538 serogroup B and all 87 serogroup C strains isolated between 1996 and 1998 were investigated by PCR with the arbitrary primer D8635, which is able to identify lineage III strains. In all, 399 strains (64%) were attributed to lineage III on the basis of PCR-based typing. Since their introduction in the Belgian population in the early 1990s, lineage III strains have become increasingly variable in phenotype. Currently, they are represented by strains belonging to 38 different phenotypes, of which 25 were not found in the period 1990-1995. The 87 serogroup C strains were further investigated by pulsed-field gel electrophoresis (PFGE), and a subset of 30 strains was also investigated by multilocus sequence typing (MLST). Strains of phenotype C:2b:P1.5,2, which currently constitute the majority of the serogroup C strains, were demonstrated to belong to cluster A4. Comparison of the discriminatory ability of D8635-primed PCR, PFGE and MLST revealed that D8635-primed PCR was the least discriminatory method and PFGE the most discriminatory method. However, the MLST data were more readily interpreted than the PFGE fingerprint patterns and can be compared easily with data obtained in other studies. In conclusion, the ongoing increase of meningococcal disease in Belgium could be attributed not only to the further expansion of lineage III, but also to the introduction of C:2b:P1.5,2 strains of cluster A4 in to the Belgian population.

Macrolide resistance and erythromycin resistance determinants among Belgian Streptococcus pyogenes and Streptococcus pneumoniae isolates.

Resistance of streptococci to macrolide antibiotics is caused by target-site modification or drug efflux. The phenotypic expression of target-site modification can be inducible or constitutive. The prevalence of the three phenotypes among Belgian erythromycin-resistant Group A streptococci (GAS) and Streptococcus pneumoniae isolates was surveyed, their MICs for seven antibiotics were determined and the clonality of the isolates was explored. Of the 2014 GAS isolates tested 131(6.5%) were erythromycin resistant (MIC > 1 mg/L): 110 (84.0%) showed the M-resistance phenotype whereas the remaining 21 strains (16.0%) were constitutively resistant. No inducibly resistant strains were detected. Of 100 S. pneumoniae isolates, 33 were erythromycin resistant (MIC > 1 mg/L). In contrast to the GAS isolates, only 9.1% of the 33 erythromycin-resistant S. pneumoniae isolates showed the M-resistance phenotype. The presence of mefA/E and ermB genes in the M-resistant and constitutively and inducibly resistant strains, respectively, was confirmed by PCR analysis. Genomic analysis based on pulsed-field gel electrophoresis (PFGE) using the restriction enzyme SfiI, revealed 54 different PFGE patterns among the 131 erythromycin-resistant GAS isolates, of which an M6 clone represented 16.0% of the strains; all other clones, exhibiting different M-types, represented <7% of the strains. The S. pneumoniae isolates also appeared to be polyclonally based, as determined by arbitrarily primed PCR. The macrolides miocamycin and rovamycin, the lincosamide clindamycin and the ketolide HMR 3647 showed excellent activity against the M-resistant GAS and S. pneumoniae strains.

Prevalence and molecular epidemiology of glycopeptide-resistant enterococci in Belgian renal dialysis units.

The molecular epidemiology of glycopeptide-resistant enterococci (GRE) colonizing the intestinal tracts of Belgian renal dialysis patients was studied among 1318 patients of a population of 1800 dialysis patients from 29 dialysis centers. Of these, 185 patients (14.0%) were colonized with a VANA-positive GRE; GRE harboring the VANB gene were not detected. The majority of the VANA GRE (80.5%) were identified as Enterococcus faecium; 14.8% were identified as E. faecalis; and a limited number were identified as E. avium, E. casseliflavus, E. dispar, E. durans, or E. gallinarum. Genome analysis of 277 VANA-positive GRE by pulsed-field gel electrophoresis revealed a high genetic variability both within the different dialysis centers and within the patients' own GRE flora. No high-level gentamicin-resistant VANA-positive GRE were detected, and most strains remained susceptible to ampicillin. These findings do not support a hospital-driven endemicity of VANA-positive enterococcal isolates in Belgium.

Comparison of glycopeptide-resistant Enterococcus faecium isolates and glycopeptide resistance genes of human and animal origins.

One hundred thirty-two glycopeptide-resistant Enterococcus faecium (GREF) isolates from different hospitals and pig and poultry farms in Belgium were compared on the basis of (i) their antibiotic susceptibilities, (ii) their SmaI pulsed-field gel electrophoresis (PFGE) patterns, and (iii) the organization of their Tn1546 or related elements in order to detect possible phenotypic and genotypic relationships among both groups of isolates. Human and animal vanA-positive GREF isolates were found to have similar susceptibility patterns; they remained susceptible to gentamicin and were, in general, susceptible to ampicillin. PFGE demonstrated a very high degree of genomic heterogeneity in both groups of isolates. However, indistinguishable isolates were found within different farms or hospitals, and in two instances, epidemiologically unrelated pig and human isolates showed indistinguishable PFGE patterns. In total, eight different transposon types were identified, and all were related to the prototype transposon Tn1546. The two predominant types, Tn1546 and type 2 transposons, which differed at three band positions, were present in both human and animal isolates. Type 2 transposons were significantly associated with pig isolates. The other types were seldom detected. These data suggest a possible exchange of glycopeptide resistance markers between animals and humans.

Biological activity and three-dimensional structure of an agonist analog of bombesin.

JMV635, a nonapeptide analog of the active terminal nonapeptide segment of bombesin, was tested for its ability to stimulate in vitro amylase release from rat pancreatic acinar cells and to inhibit the binding of gastrin-releasing peptide to rat pancreatic acini. It was found to be a full agonist of bombesin and to recognize the bombesin receptor with moderate potency. The NMR proton assignments of JMV635 were achieved, and the conformations of JMV635 in aqueous solution and in trifluoroethanol at 297 K were determined using two-dimensional COSY, HOHAHA, NOESY and ROESY experiments. In trifluoroethanol, JMV635, like the active part of bombesin, showed a partial alpha-helical structure. These results were confirmed by circular dichroism and refined by restrained molecular dynamic methods. Structure calculations, using the distance and angle restraints obtained from NMR data on JMV635, gave a total of 75 structures which could be aligned to a root mean square deviation of the bond length of 0.007 A and of the valence angle of 1.55 degrees for the backbone atoms of the amino acid residues. The conformation is a well-defined right-handed alpha-helix in the C-terminal Q2-G6 segment and is less structured in the three C-terminal residues.

[Astragalocalcanean subluxation in children]. / La subluxation astragalo-calcanéenne chez l'enfant.

The authors report a unique case of talo-calcaneal subluxation in a child following minor trauma. The reduction proved to be straightforward, and the outcome was entirely favorable.

A quantitative map of nucleotide substitution rates in bacterial rRNA.

A recently developed method for estimating the variability of nucleotide sites in a sequence alignment [Van de Peer, Y., Van der Auwera, G. and De Wachter, R. (1996) J. Mol. Evol. 42, 201-210] was applied to bacterial 16S, 5S and 23S rRNAs. In this method, the variability of each nucleotide site is defined as its evolutionary rate relative to the average evolutionary rate of all the nucleotide sites of the molecule. Spectra of evolutionary rates were calculated for each rRNA and show the fastest evolving sites substituting at rates more than 1000 times that of the slowest ones. Variability maps are presented for each rRNA, consisting of secondary structure models where the variability of each nucleotide site is indicated by means of a colored dot. The maps can be interpreted in terms of higher order structure, function and evolution of the molecules and facilitate the selection of areas suitable for the design of PCR primers and hybridization probes. Variability measurement is also important for the precise estimation of evolutionary distances and the inference of phylogenetic trees.

Specific detection of Campylobacter concisus by PCR amplification of 23S rDNA areas.

The phenotypic detection of Campylobacter concisus, a species of considerable genomic and phenotypic heterogeneity, has proven to be rather tedious in the past. Although alternative methods like DNA:DNA hybridization, immunotyping or whole-cell protein electrophoresis are valuable for the specific detection of C. concisus, they are too laborious to be performed in routine settings. Hence a simple Campylobacter concisus-specific PCR assay was developed, based on a target sequence which comprises the most variable areas of 23S rDNA. The PCR assay was successfully evaluated on a broad selection of C. concisus strains and phylogenetically related bacteria.

Database on the structure of large ribosomal subunit RNA.

A database on large ribosomal subunit RNA is made available. It contains 258 sequences. It provides sequence, alignment and secondary structure information in computer-readable formats. Files can be obtained using ftp.

Structure of the large ribosomal subunit RNA of Phytophthora megasperma, and phylogeny of the oomycetes.

The 5.8S and 28S rRNA sequences of the oomycete Phytophthora megasperma were determined in order to study the secondary structure of these molecules and to assess the phylogenetic position of the oomycetes among the eukaryotes. Preliminary results point to an affiliation between the oomycetes, dinoflagellates and ciliates, a cluster which seems related to the fungi. In the course of this work, we developed a set of primers which allow sequencing and PCR amplification of eukaryotic large ribosomal subunit RNA genes of a wide range of phylogenetically distant organisms.

Discrimination among thermophilic Campylobacter species by polymerase chain reaction amplification of 23S rRNA gene fragments.

By comparing nucleic acid sequences determined for one of the most variable areas of 23S rRNA genes of 23 Campylobacter strains, we were able to identify regions specific for thermophilic Campylobacter strains. Oligonucleotide primers corresponding to these unique regions were synthesized and used in the polymerase chain reaction. One primer pair selectively detected all thermophilic Campylobacter species, while four other primer pairs allowed discrimination among the thermophilic species Campylobacter coli, Campylobacter jejuni subsp. jejuni, Campylobacter lari, and Campylobacter upsaliensis. All primer sets were tested successfully on a large number of clinical isolates.

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences.

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence for Campylobacter jejuni subsp. jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.

Compilation of small ribosomal subunit RNA structures.

The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk.

Plasmalogens and O-alkylglycerophospholipids in aquatic animals.

1. Qualitative and quantitative distribution of ether phospholipids was studied in several aquatic organisms. 2. Brain, nerve and gills of these animals were rich in ether glycerophospholipids. 3. 1-alkenyl ethers were in general more abundant than alkyl ones. The alkenyl ethers were mainly components of PE in all aquatic animals. Those of PS occurred in mollusca only. 4. Alkyl ether moieties appeared to be essentially located in PE and PC. 5. This review suggests that ether phospholipids may play an important role in the permeability properties of biological membranes.

Phospholipid composition and metabolism of crustacean gills as related to changes in environmental salinities: relationship between Na+-K+-ATPase activity and phospholipids.

The phospholipid composition and metabolism are studied in crustacean gills. It is reported that branchiae are rich in PC, PE and DPG and abundant in long chain polyunsaturated fatty acids (20:4 omega 6 and 20:5 omega 3 acids). The pathways of phospholipids synthesis appear similar to those described for vertebrates. It is demonstrated that there exist significant differences in the level of phosphatides between the anterior and posterior gills of Eriocheir sinensis. No matter what the salinity, the three more posterior located gills of Chinese crabs are shown to contain much more unsaturated phospholipids (PE and DPG). This is particularly true when animals are acclimated to dilute media. Moreover, lipids of posterior gills appear more fluid than the anterior ones as reported by the values of the degree of fluorescence polarization and the index of unsaturation of fatty acids. It is suggested that these lipid changes may indicate the existence of a functional difference between the various branchiae of euryhaline Eriocheir sinensis with respect to their ability to transport salt. It is shown that the renewal of DPG and PS is increased in posterior gills isolated from freshwater Chinese crabs. It is postulated that the enhanced formation of DPG in posterior gills is an indicator of an increased synthesis of mitochondria having as principal function to produce the necessary energy for the Na+ uptake. An attempt is made to correlate the PS metabolism and the Na+-K+-ATPase activity which is particularly located in the mitochondrial fractions of the three pairs of posterior gills.

Phospholipids and cholesterol in brush border and basolateral membranes from rat intestinal mucosa.

Total phospholipids and free cholesterol reach higher levels in basolateral membranes than in brush border membranes, these membranes being purified from rat intestinal mucosa. Cholesterol/phospholipid and cholesterol/phospholipid choline molar ratios are lower in basolateral membranes. Lysophosphatidylcholine, sphingomyelin and phosphatidylinositol are approximately twice as high in brush border membranes (respectively, 8.7, 24.1 and 22.3% total lipid phosphorus) when compared to basolateral membranes (respectively, 3.6, 12.5 and 11.5% total lipid phosphorus). Conversely, basolateral membranes are richer than brush border membranes in phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and diphosphatidylglycerol (basolateral membranes: 36.9, 8.1, 24.8, 1.7% total lipid phosphorus; brush border membranes: 26.7, 4.4, 11.9, 0.5% total lipid phosphorus.