Localized conversion in Streptococcus pneumoniae recombination: heteroduplex preference.

In pneumococcal transformation the frequency of recombinants between point mutations is generally proportional to distance. We have recently described an aberrant marker in the amiA locus that appeared to enhance recombination frequency when crossed with any other allele of this gene. The hyperrecombination that we have observed in two-point crosses could be explained by two hypotheses: the aberrant marker induces frequent crossovers in its vicinity or the mutant is converted to wild type. In this report we present evidence showing that, in suitable three-point crosses, this hyperrecombination does not modify the recombination frequency between outside markers, suggesting that a conversion occurs at the site of this mutation. To estimate the length over which this event occurs, we isolated very closely linked markers and used them in two-point crosses. It appears that the conversion system removes only a few base pairs (from three to 27) around the aberrant marker. This conversion process is quite different from the mismatch-repair system controlled by hex genes in pneumococcus, which involves several thousand base pairs. Moreover, we have constructed artificial heteroduplexes using separated DNA strands. It appears that only one of the two heteroduplexes is specifically converted. The conversion system acts upon 5'..ATTAAT..3'/3'.. TAAGTA..5'. A possible role of the palindrome resulting from the mutation is discussed.

Conversion of deletions during recombination in pneumococcal transformation.

Genetic analysis of 16 deletions obtained in the amiA locus of pneumococcus is described. When present on donor DNA, all deletions increased drastically the frequency of wild-type recombinants in two-point crosses. This effect was maximal for deletions longer than 200 bases. It was reduced for heterologies shorter than 76 bases and did not exist for very short deletions. In three-point crosses in which the deletion was localized between two point mutations, we demonstrated that this excess of wild-type recombinants was the result of a genetic conversion. This conversion extended over several scores of bases outside the deletion. Conversion takes place during the heteroduplex stage of recombination. Therefore, in pneumococcal transformation, long heterologies participated in this heteroduplex configuration. As this conversion did not require an active DNA polymerase A gene it is proposed that the mechanism of conversion is not a DNA repair synthesis but involves breakage and ligation between DNA molecules. Conversion of deletions did not require the Hex system of correction of mismatched bases. It differs also from localized conversion. It appears that it is a process that evolved to correct errors of replication which lead to long heterologies and which are not eliminated by other systems.

Conversion of large heterologies in Streptococcus pneumoniae.

In genetic transformation, long deletions dramatically increase the frequency of wild-type recombinants in 2-point crosses. In 3-point crosses in which the deletion was localized between 2 point mutations we demonstrated that this hyper-recombination was the result of genetic conversion extending over several scores of bases outside the deletion. As this conversion did not require an active DNA polymerase A gene, it was proposed that the mechanism of conversion involves breakage and ligation between DNA molecules. A similar hyper-recombination was observed when donor DNA carried an insertion. These results suggest that long heterologies participated in recombination so that surrounding homologous regions are almost completely paired and that these long heterologies are converted. It appears that it is a process that evolved to correct errors of replication which lead to long deletions and which are not eliminated by other systems.

Long- and short-patch gene conversions in Streptococcus pneumoniae transformation.

In pneumococcal transformation some point mutations are integrated by an excision-repair pathway which switches the heteroduplex DNA into homoduplex. This transfer of information is a gene conversion. We have reviewed some of the properties of this system especially those relating to heteroduplex specificity and given evidence that this extends over several kilobases of DNA. We then describe a new process of conversion in pneumococcal transformation which occurs over a very short distance (5 to 27 base-pairs) and is triggered by a single site mutation resulting from the transversion 5'-ATTCAT...to 5'...ATTAAT... Only one of the two heteroduplexes 5'...A...3'/3'...G...5', is converted.

Preferential redistribution of lipoprotein-unassociated apoA-IV to an HDL subpopulation with a high degree of LCAT modification.

The relationship between LCAT mediated HDL modification and the redistribution of lipoprotein-unassociated apoA-IV to HDL was investigated in vitro. Immunoaffinity-isolated rat lipoprotein-unassociated apoA-IV was added to apoB-, apoE-, apoA-IV depleted, [3H]cholesterol labelled rat plasma and incubated at 37 degrees C. The addition of lipoprotein-unassociated apoA-IV resulted in a modest (10%) but significant reduction in the rate of cholesterol esterification. Incubations conducted in the presence of active LCAT led to a time-dependent increase in the amount of the 3H label retained by an anti-apoA-IV immunoaffinity column. Lipoproteins retained by the anti-apoA-IV immunoaffinity column had experienced a greater conversion of [3H]cholesterol to [3H]cholesteryl esters (48% esterification at 30 min) than the unretained lipoproteins (19% esterification at 30 min). These data suggest that during the course of LCAT-induced cholesterol esterification, lipoprotein-unassociated apoA-IV transfers to a subpopulation of HDL which has been modified by LCAT to a greater extent than the remaining HDL. Further analysis of the data demonstrates that 48% cholesterol esterification is sufficient to allow apoA-IV to be accommodated on the surface of an HDL particle.

[Gardnerella vaginalis. Frequency of its isolation from women complaining of leukorrhea]. / Gardnerella vaginalis. Fréquence de son isolement chez la femme se plaignant de leucorrhées.

A study was carried out on 131 vaginal swabs that were taken from 98 women who had a discharge and 33 women who were asymptomatic to determine the frequency in which Gardnerella vaginalis was discovered. This study shows that this bacteria is the only micro-organism that is found in a significantly higher number of women with nonspecific discharges (37%) than in those who have no discharge (9.1%). These results, while they do not show that Gardnerella vaginalis is the only aetiological factor in nonspecific vaginitis, do underline that it is a factor in thinking about such a syndrome and therefore it should be looked for by the laboratory and by the doctor treating patients.

[Etiology of bacterial vaginosis (non-specific vaginitis)]. / Etiologie de la vaginose bactérienne (vaginite non spécifique).

56 women who were diagnosed bioclinically as having a bacterial vaginal infection were studied, as were 35 women as a control group. The study was a semi-quantitative analysis of the vaginal bacterial flora, both aerobic and anaerobic. It shows that Gardnerella vaginalis and anaerobic bacteria such as Peptococcus, Peptostreptococcus, Bacteroïdes, Veillonella and Mobiluncus were associated in a statistically significant way with bacterial vaginitis. On the other hand Lactobacilli were less frequently found (p less than 0.001) than in the control group of women. The way in which the microbial flora is changed has been observed during attacks of vaginitis and is discussed, as is the importance of making the diagnosis and of treating this syndrome.

[Calculation of the blood volume to be removed for intentional normovolemic hemodilution]. / Règle à calcul du volume sanguin à prélever pour réaliser une hémodilution normovolémique intentionnelle.

Intentional haemodilution is more and more frequently used. Although it is a simple technique, it can only be carried out with a maximum of safety if the operator has an idea of the amount of blood that needs to be removed to obtain the required haematocrit value. Several methods have been suggested, using more or less complex calculations, or nomogrammes. A new simple rule is presented here which gives an estimation of the amount of blood to be removed in three steps: 1) the theoretical total blood volume (VS), according to weight, height and sex of the patient, 2) the ratio (R) between the initial and required final haematocrit, and 3) the volume of blood to be removed (Vp) according to VS and R. A mono-compartment model with identical inflow and outflow was used. A multicentre trial with 229 haemodilutions showed that the volume removed (mean = 1,325 ml; SD = 642 ml) was 15% less than the calculated volume (mean = 1,526 ml; SD = 561 ml). No one factor was found to explain this difference. The haematocrit values obtained by microcentrifuge were compared with those obtained by a laboratory automatic counter. The mean of the differences was 0.002 (SD = 0.029). Because of the possible errors involved in estimating the total blood volume and the haematocrit before dilution, it is essential that the haematocrit be checked at least once during the haemodilution.

[Bacteria isolated in 1994-1995 in female upper genitalia infections and in male urethritis. Distribution and sensitivity to antibiotics]. / Bactéries isolées en 1994-1995 au cours des infections gynécologiques hautes et des uréthrites masculines. Distribution et sensibilité aux antibiotiques.

OBJECTIVE: Classify antibiotics according to their individual activity so as to identify those suitable for empiric therapy. METHODS: We studied bacterial strains isolated from patients with urethritis (n = 189) and upper genital tract infections (n = 163) between June 1994 and February 1995 in 3 hospital and 4 community laboratories. Upper genital tract infections were divided into two groups: proven infection on laparoscopy specimen (n = 79) and suspected infection with isolation of pathogen in cervical samples (n = 84). Pathogens isolated were: Chlamydia trachomatis in 36/12/15 cases respectively, Mycoplasma hominis in 12/20/13, Ureaplasma urealyticum in 55/30/15, Neisseria gonorrhoeae in 40/2/0, Haemophilus spp in 20/2/1, group B streptococci in 7/1/8, E. coli in 8/1/17 and miscellaneous in 11/8/15. The minimal inhibitory concentrations for all strains were determined in 4 laboratories for ofloxacin, erythromycin and doxycyclin against C. trachomatis, M. hominis and U. urealyticum, and for ofloxacin, erythromycin, doxycyclin, amoxicillin+clavulanate, cefotaxime and gentamicin against the other strains. The activity score (% susceptibility to each antibiotic weighted by the frequencies of each isolate in urethritis and upper genital tract infection based on recent French epidemiologic data) was calculated for each antibiotic. CONCLUSION: The antibiotics with the best empiric activity scores in urethritis were, in decreasing order: doxycyclin (90.4%), ofloxacin (88.1%), and erythromycin (50.2%). The most active combinations in upper genital tract infections were ofloxacin+amoxicillin (100%), doxycyclin+cefotaxime+metronidazole (95.9%) and doxycyclin+amoxicillin (95.3%).

[Quantitative determination of FTA abs IgM in different stages of syphilis before and after treatment]. / Apport du FTA abs IgM uantitatif aux différents stades de la syphilis avant et après traitement.

The titres of treponemal specific IgM and IgG antibodies were determined by monospecific immunofluorescence (FTA abs IgM/IgG) on 191 sera from 107 patients with treated or untreated syphilis, at various stages and from 10 neonates born from seropositive mothers. IgM antitreponemal antibody was found in all cases of untreated syphilis but two neurosyphilis. After treatment the antibody usually disappeared within one year. However it persisted in three patients treated two years ago or more. When sera from these patients were fractionated by density gradient ultracentrifugation and examined for antitreponemal antibodies of the IgM classe (FTA abs IgM 19S) they never showed detectable concentrations of IgM. False reactive results as false non reactivity due to competitive inhibition, observed with FTA abs IgM can be eliminated after separation of immunoglobulins.

[Syphilitic labial leucokeratosis]. / Leucokératose labiale syphilitique.

BACKGROUND: Several diagnoses, including syphilis, can be entertained in patients with leukokeratosis of the buccal mucosa. We report a case of labial leucokeratosis which revealed latent syphilis. CASE REPORT: A 36-year-old man with a past history of genital syphilis chancre which have been treated 12 years earlier, developed buccal leucokeratosis with no other clinical manifestation. Histology showed dermal infiltration containing plasma cells, polynuclears and lymphocytes. Blood tests were positive for syphilis. Complementary examinations were unable to detect another localization. Leucokeratosis regressed completely after one injection of Extencilline. There has been no recurrence at one year. DISCUSSION: The clinical and histological presentations of syphilis can mimic different skin diseases. Serodiagnosis alone is significant. Isolated buccal lesions are rarely described in syphilis suggesting serodiagnosis should always be ordered. Whatever the clinical stage of the diseases, serological surveillance after treatment for syphilis is essential.

[Recent bacteriologic data: from physiopathology to treatment]. / Données bactériologiques récentes: de la physiopathologie au traitement.

Bacterial vaginosis is characterized by a change in the vaginal ecosystem in which Lactobacillus spp, the dominant members of the normal flora, are replaced by an association of various bacterial species including Gardnerella vaginalis, anaerobes (Bacteroides spp, Prevotella spp, Porphyromonas spp, Peptostreptococcus spp and Mobiluncus spp) and Mycoplasma hominis. The reasons for this imbalance are unknown, although the loss of lactobacilli that produce hydrogen peroxide (which is toxic for G. vaginalis and numerous anaerobes) may be an essential element. The introduction of one or several of these species into the vagina (they can occasionally be isolated in small numbers even in the absence of vaginosis) following intercourse or from the intestinal tract may also play a role. G. vaginalis itself is not considered to cause vaginosis, but is almost always present and multiplies rapidly; in addition, it has a high capacity for adherence to epithelial cells both in vivo and in vitro. It is now agreed that the concomitant growth of one or several anaerobic species is required for bacterial vaginosis to develop. Our knowledge of the bacteriology of bacterial vaginosis has implications for diagnosis and treatment. It is now possible to obtain a precise bacteriologic diagnosis, not by culturing G. vaginalis (a costly and low-yield procedure), but by direct examination of the vaginal flora after Gram staining, which shows the replacement of lactobacilli by a characteristic polymorphic flora. Therapy is based on the use of antibiotics such as the imidazoles, which are active against G. vaginalis and anaerobes, but not against the commensal lactobacilli.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative in vitro susceptibility of a tetracycline-resistant Chlamydia trachomatis strain isolated in Toulouse (France).

BACKGROUND AND OBJECTIVES: We recently reported the first isolation of a tetracycline-resistant Chlamydia trachomatis strain in Toulouse from a woman treated with tetracycline. To characterize this isolate, its in vitro susceptibility was compared with those of 34 other C. trachomatis isolates recovered in Toulouse. STUDY DESIGN: The susceptibilities of C. trachomatis strains were determined in terms of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using McCoy cells in 96-well microdilution plates, with an inoculum of 5.10(3) to 1.10(4) inclusion-forming units/ml. The antimicrobial agents tested were tetracycline, azithromycin, erythromycin, ofloxacin, and pristinamycin. RESULTS: No difference was observed between the MICs and MBCs except for the tetracycline. Tetracycline-resistant strain MIC and MBC were > 64 micrograms/ml, although < 1% of the bacterial population showed resistance. For the other isolates, the MIC of tetracycline was < or = 0.25 microgram/ml. The antibiotics other than tetracycline were active in vitro against all strains. CONCLUSIONS: These results show that the tetracycline resistance observed in Toulouse differs from the "heterotypic resistance" described previously in the United States in multiresistant C. trachomatis isolates. They confirm that the resistance we observed may be a new phenomenon.

Comparative in vitro activities of pristinamycin and other antimicrobial agents against genital pathogens.

The MICs of pristinamycin for genital pathogens were compared with those of ampicillin, tetracycline, erythromycin, and ciprofloxacin. Pristinamycin was active against all the strains studied. Because of this activity and its lack of toxicity, pristinamycin might be a valuable therapeutic agent for treating major sexually transmitted diseases.

[First isolation in France of a penicillinase-non producing Neisseria gonorrhoeae with high level tetracycline resistance]. / Premier isolement en France d'une souche de Neisseria gonorrhoeae non productrice de pénicillinase présentant un haut niveau de résistance à la tétracycline.

We report the first isolation in France of a penicillinase-non producing Neisseria gonorrhoeae with high level tetracycline resistance. The minimal inhibitory concentrations (MICs) of nine antibiotics were determined by agar dilution for the tetracycline resistant strain and 39 other penicillinase-non producing N. gonorrhoeae strains which were isolated during the same year (1989), in Toulouse. No difference was observed between the MICs except for the tetracycline (tetracycline-resistant strain MIC = 32 mg/l). The commonly used antibiotics other than tetracycline were active in vitro against all the strains. The plasmid content of the tetracycline-resistant strain was analysed by agarose gel electrophoresis and revealed the presence of two plasmids: the 2.6-megadalton in cryptic plasmid and the 25.2-megadalton Tet M conjugative plasmid.

Study of the in vitro activity of new cephalosporins on strains of Neisseria gonorrhoeae of the Toulouse region.

The study was carried out with 50 strains of gonococci. Susceptibility to the antibiotics was determined by standard sensitivity test, by systematic test for beta-lactamase and by measurement of the MICs. The cephalosporins tested were: moxalactam, cefotaxime, ceftriaxone, cefotiam, cefoxitin, cefazolin and cephalothin. Their activities were also compared with other beta-lactams, namely penicillin and ampicillin. The efficacy in vitro of the cephalosporins: in the lead, ceftriaxone, remarkably active, with cefotaxime following very close, and then moxalactam with cefotiam a little behind, and finally all the other beta-lactams tested. The median MICs of the new cephalosporins (moxalactam, cefotaxime, ceftriaxone and cefotiam) were at least 10 times greater than those of the other beta-lactams tested. The MIC values observed with a resistant strain (penicillin, ampicillin; MIC 256 microgram/ml) never exceed the maximum values found with the 50 susceptible strains.

[Isolation and characterization of a beta-lactamase-specifying plasmid in a strain of "neisseria gonorrhoeae" (author's transl)]. / Isolement et caractérisation d'un plasmide de résistance à la pénicilline, dans une souche de Neisseria gonorrhoeae.

GCL3, a beta-lactamase-producing, penicillin resistant strain of Neisseria gonorrhoeae isolated in Toulouse (France), and GCL51, a penicillin susceptible strain, were examined for their plasmid content. Agarose-gel electrophoresis following or not ultracentrifugation in a cesium chloride-ethidium bromide gradients, revealed, for both strains a 3.9-kilobase (kb) (2.6 megadaltons) cryptic plasmid. Penicillin resistant strain GCL3 also contained a 37 kb (24.5 megadaltons) and a 7.3 kb (4.8 megadaltons) plasmid. Transformation studies showed that the gene responsible for beta-lactamase production was carried by the 7.3-kb plasmid. E. coli cells transformed for ampicillin resistance by plasmid DNA from GCL3, contained a single 7.3 kb-plasmid. The restriction endonuclease cleavage map obtained for this plasmid indicated that it is closely related to a penicillin R plasmid previously described in a strain isolated in the USA. The first isolation in the Midi-Pyrenees area of a beta-lactamase-producing strain proved the necessity of a rigorous surveillance of N. gonorrhoeae strains isolated in the world.