Mobilization of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 into Escherichia coli by cointegration with several gram-conjugative plasmids.

We report the mobilization by cointegration of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 in an Escherichia coli background. Transfer of pSJ5.2 was measured by filter mating assays with five different conjugative plasmids from Enterobacteriaceae and the gonococcal 41 kb tet(M). Plasmid pSJ5.2 was mobilized to E. coli at frequencies of 1.7x10(-6), 9.3x10(-8) and 2.7x10(-5) by the tet(M), R64 drd-33 and N3 conjugative plasmids, respectively. Mobilization of pSJ5.2 by the 41 kb tet(M) conjugative plasmid resulted in stable Amp(R) E. coli transconjugants consisting of pSJ5.2 plasmid with an insertion located in the 2.4 kb BamHI-BamHI fragment. Mobilization of pSJ5.2 by R64drd-33 and N3 conjugative plasmids involved stable cointegrates as detected by Southern Blot with a DIG-labelled PstI-digested pSJ5.2 probe. Restriction analysis of the R64::pSJ5.2 and N3::pSJ5.2 cointegrates and Southern Blot with the pSJ5.2 probe showed that cointegrates formed by deletion of DNA regions within the 1.8 kb BamHI-HindIII fragment of pSJ5.2. The plasmid thus appears to use multiple recombination mechanisms for cointegration with different conjugative plasmids. The complete nucleotide sequence of pSJ5.2 was determined, and will be a useful tool to further investigate the molecular mechanisms leading to its cointegrative transfer.

Vaccine evaluation studies of replication-defective SIVsmB7.

Non-infectious virus-like particles of SIVsmB7 that expresses env and gag gene products but are defective in pol and vpx/vpr were assessed for their ability to induce protective immunity against infection with pathogenic SIVsmE660 in rhesus macaques. Animals were immunized in three groups: group A was primed with cell-associated SIVsmB7 and boosted with cell-free SIVsmB7; group B was primed with cell-free SIVsmB7 and boosted with cell-free SIVsmB7 conjugated to iron oxide microbeads; group C was primed with cell-free SIVsmB7 mixed with Titer Max adjuvant and boosted with cell-free SIVsmB7 mixed with SAF-M adjuvant followed by secondary boosting with cell-free SIVsmB7 conjugated to microbeads. Animals were challenged intravenously with 20 animal infectious doses of SIVsmE660 grown in rhesus peripheral blood mononuclear cells 3 weeks after final boosting. All animals became infected as evidenced by quantitative virus cultivation. Sera from immunized animals contained low-titer antibodies by ELISA and low or undetectable neutralizing antibodies on the day of challenge but strong anamnestic antibody responses were observed following challenge. Interestingly, 2 of 3 animals in group A showed evidence of transient viremia and more stable CD4 counts following challenge as compared to the other immunized animals and to non-immunized controls. Thus, immunization with cell-associated SIVsmB7 did not provide sterilizing immunity against challenge with a highly pathogenic SIV strain but might have caused virus clearance later in infection.

Effect of penicillin and spectinomycin given for urethritis and cervicitis with Neisseria gonorrhoeae: high prevalence of penicillin-resistant isolates.

Efficacy of single-dose spectinomycin (TRO: 2 g intramuscularly) was compared with that of aqueous procaine penicillin G (APPG:4.8 x 10(6) units) plus 1 g of probenecid for treatment of gonococcal urethritis and cervicitis. Cure rates of the 210 patients who received TRO and 190 patients who received APPG were 97.6% and 91.1%, respectively. MICs of antibiotics were determined using the agar dilution method. Those isolates with MICs of APPG of less than 1.0 microgram/ml had low failure rates (2.9%), while strains with increased resistance to APPG (MICs greater than or equal to 1.0 microgram/ml) had higher failure rates (24%). Treatment failures seen with TRO were not correlated to isolates with the higher MICs. Clinical results suggest TRO could be given for treatment of genital gonorrhoea in Puerto Rico due to the high prevalence of both chromosomally-mediated penicillin-resistant Neisseria gonorrhoeae (20%) and penicillinase-producing Neisseria gonorrhoeae (7.5%) strains and the high rate of failure seen with the use of APPG.

Interactions of HIV and STDs in a group of female prostitutes.

PIP: Incidence of HIV and of several other sexually transmitted diseases (STDs) was determined in 171 female prostitutes from 3 sites in San Juan, Puerto Rico. The sites were selected by high incidence of penicillinase-producing N. gonorrhoeae in clients of prostitutes. These women came from about a dozen different countries, mostly Latin American. 14% reported they always used condoms. Specimens were taken of blood, endocervix, cervix, rectum and oropharynx, and tested for HIV, gonorrhea, syphilis, herpes, chlamydia, hepatitis B and cytomegalovirus. 18% harbored gonorrhea, of which 13% were penicillinase positive. Syphilis occurred in 8%. Chlamydia was the most prevalent infection, in 47% of subjects. Serological evidence of hepatitis B was apparent in 53%, and of cytomegalovirus in 99%. HIV status was tested after unlinking identifying information from 80 serum samples, and 16% were confirmed HIV positive. Women from the site frequented by more street walkers than bar girls had a higher incidence of hepatitis B, and were known to be more frequent users of IV drugs. These data confirm observations made elsewhere that HIV infection may coexist with other STDs.^ieng

Molecular and cellular events during the yeast to mycelium transition in Sporothrix schenckii.

Unbudded singlets from exponentially growing yeast cells of Sporothrix schenckii were harvested, selected by filtration and allowed to form germ tubes in a basal medium with glucose at pH 4.0 and 25 degrees C. These conditions supported only the development of the mycelial form of S. schenckii in a reproducible manner which allowed further analysis of the early cellular events occurring during the yeast-to-mycelium transition. The relationship between macromolecular synthesis (DNA and RNA synthesis) and nuclear division, hyphal growth and septum formation were investigated during germ tube formation. RNA synthesis started 0 to 3 h after the induction of germ tube formation, followed by DNA synthesis and the first nuclear division, which took place between 3 and 6 h. Germ tube formation followed nuclear division and was first evidenced 6 h after the induction of germ tube formation, but was not completed until 12 h after inoculation. Septation was first observed in these germ tubes at the mother cell-germ tube junction 6 h after induction. Addition of hydroxyurea, an inhibitor of DNA synthesis, to the medium, also inhibited nuclear division and germ tube growth, suggesting that these processes in S. schenckii are dependent upon DNA synthesis.

DNA content, kinetic complexity, and the ploidy question in Candida albicans.

Candida albicans is a dimorphic fungus that is pathogenic for humans. No sexual cycle has been reported for this fungus, and earlier reports have differed on whether typical strains of C. albicans are haploid or diploid. Previous estimates of the DNA content of C. albicans varied by one order of magnitude. We used three independent methods to measure the kinetic complexity of the single-copy DNA from a typical strain of C. albicans (strain H317) to determine the DNA content per haploid genote; we obtained values of 15 and 20 fg per cell by using S1 nuclease and hydroxyapatite assays, respectively. Optical assays for DNA reassociation kinetics, although not definitive in themselves, yielded values in this range. Chemical measurements of the DNA content of several typical strains, including strain H317, yielded values clustered about a mean of 37 fg per cell. We concluded that these strains are diploid.

Protoplasts from yeast and mycelial forms of Candida albicans.

Protoplasts have been obtained in high yields from the yeast and mycelial forms of a variety of strains of Candida albicans by enzyme digestion of cells with commercially available lytic enzymes. The protoplast formation procedure was equally effective for exponential and stationary phase cells. Pretreatment with dithiothreitol and Pronase in the presence of EDTA and Tris was necessary. Other thiol reagents and conditions did not release protoplasts from all the strains of C. albicans tested. Treatment with digestive juice of the snail Helix pomatia required the addition of chitinase for the release of protoplasts from most strains tested. Conditions for maximizing the yield of protoplasts and the activities of beta-glucuronidase and chitinase were determined. Electron microscopy of C. albicans showed that the pretreatment conditions removed the outer layers and the treatment itself completely removed the inner layers of the cell wall. More than 90% of the protoplasts produced by this model were viable as assessed by vital staining with Janus Green B.