Gardnerella vaginalis population dynamics in bacterial vaginosis.

Bacterial vaginosis (BV) is the leading cause of vaginal discharge and is associated with the facultative Gram-variable bacterium Gardnerella vaginalis, whose population structure consists of four clades. Our goal was to determine if these clades differ with regard to abundance during BV. We performed a short-term longitudinal study of BV. Patients were evaluated according to the Amsel criteria and Nugent scoring at initial diagnosis, immediately after treatment and at a 40- to 45-day follow-up visit. G. vaginalis clade abundance was determined by quantitative real-time polymerase chain reactions (qPCRs). Among all specimens, the abundance of clades 1 and 4 were higher than that of clades 2 and 3 (P < 0.001). In general, the abundance of each clade increased with the degree of vaginal dysbiosis, as determined by the Nugent score and was greater in women with Amsel 4 compared with those with Amsel 0. Only clade 1 abundance was greater when Amsel 0 or 1 specimens were compared with Amsel 2 or 3 specimens (P < 0.01). Following antimicrobial treatment, abundance of clades 1 (P < 0.001) and 4 (P < 0.05) decreased regardless of the clinical and microbiological outcome, whereas clade 2 only decreased in women who had a sustained treatment response for 40-45 days (P < 0.01). Recurrent BV was characterized by post-treatment increases of clade 1 and 2 (P < 0.01). Clades 1 and 4 predominate in vaginal specimens. Clade abundance differs with regard to the Nugent score, the Amsel criteria, and response to therapy and BV recurrence.

Vulvodynia and fungal association: a preliminary report.

Vulvodynia (vulvar pain syndrome) is a chronic multifactorial disease affecting almost 13 million women in the USA and can lead to morbidity and a reduced quality of life. We hypothesize that an initial microbiological insult in the vagina causes modifications in the biological vaginal milieu and/or an alteration on the lactobacilli flora. The vaginal milieu responds to the insult by developing an inflammatory reaction with abnormal cytokine production. These hypotheses were tested quantifying vaginal lactobacillus and cytokines, in patients with vulvodynia compared to matched healthy controls. Our preliminary data suggest a vaginal flora alteration and an immunological response involving Candida in patients with vulvodynia. Ongoing studies will assist us to clarify these findings.

Antimicrobial non-susceptibility of cervico-vaginal and rectal Escherichia coli isolates is associated with phylogeny and plasmid carriage.

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs) in adult women, which are increasingly refractory to antimicrobial treatment. UPEC colonizes the vagina prior to causing a UTI. Our hypothesis was that the vaginal flora would be enriched in UPEC and therefore have a greater prevalence of non-susceptibility relative to the rectal flora. We used disk diffusion to determine the antimicrobial susceptibilities of 100 cervico-vaginal E. coli (CVEC) and 100 rectal E. coli (REC) isolates from 200 different patients. Phylogeny, plasmid replicons, and antimicrobial resistance genes were detected by polymerase chain reaction (PCR). There were no significant differences between CVEC and REC, and the overall levels of non-susceptibility were 39.5% for ampicillin (AM), 11.5% for ampicillin-sulbactam (SAM), 11.5% for trimethoprim-sulfamethoxazole (SXT), 5% for ciprofloxacin (CIP), 2.5% for nitrofurantoin (F/M), 0.5% for ceftazidime (CAZ), 0.5% for cefotaxime (CTX), and 0% for fosfomycin (FOS). SXT non-susceptibility was associated with phylogenetic groups A and D compared with B2. AM and SXT non-susceptibility was associated with plasmid carriage. The vaginal flora is not enriched in antimicrobial non-susceptibility relative to the rectal flora. However, antimicrobial non-susceptibility was associated with phylogeny and plasmid carriage.

nimO, an Aspergillus gene related to budding yeast Dbf4, is required for DNA synthesis and mitotic checkpoint control.

The nimO predicted protein of Aspergillus nidulans is related structurally and functionally to Dbf4p, the regulatory subunit of Cdc7p kinase in budding yeast. nimOp and Dbf4p are most similar in their C-termini, which contain a PEST motif and a novel, short-looped Cys2-His2 zinc finger-like motif. DNA labelling and reciprocal shift assays using ts-lethal nimO18 mutants showed that nimO is required for initiation of DNA synthesis and for efficient progression through S phase. nimO18 mutants abrogated a cell cycle checkpoint linking S and M phases by segregating their unreplicated chromatin. This checkpoint defect did not interfere with other checkpoints monitoring spindle assembly and DNA damage (dimer lesions), but did prevent activation of a DNA replication checkpoint. The division of unreplicated chromatin was accelerated in cells lacking a component of the anaphase-promoting complex (bimEAPC1), consistent with the involvement of nimO and APC/C in separate checkpoint pathways. A nimO deletion conferred DNA synthesis and checkpoint defects similar to nimO18. Inducible nimO alleles lacking as many as 244 C-terminal amino acids supported hyphal growth, but not asexual development, when overexpressed in a ts-lethal nimO18 strain. However, the truncated alleles could not rescue a nimO deletion, indicating that the C terminus is essential and suggesting some type of interaction among nimO polypeptides.

Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.

Proteolysis and tyrosine phosphorylation of p34cdc2/cyclin B. The role of MCM2 and initiation of DNA replication to allow tyrosine phosphorylation of p34cdc2.

Previously, it has been shown that Aspergillus cells lacking the function of nimQ and the anaphase-promoting complex (APC) component bimEAPC1 enter mitosis without replicating DNA. Here nimQ is shown to encode an MCM2 homologue. Although mutation of nimQMCM2 inhibits initiation of DNA replication, a few cells do enter mitosis. Cells arrested at G1/S by lack of nimQMCM2 contain p34(cdc2)/cyclin B, but p34(cdc2) remains tyrosine dephosphorylated, even after DNA damage. However, arrest of DNA replication using hydroxyurea followed by inactivation of nimQMCM2 and bimEAPC1 does not abrogate the S phase arrest checkpoint over mitosis. nimQMCM2, likely via initiation of DNA replication, is therefore required to trigger tyrosine phosphorylation of p34(cdc2) during the G1 to S transition, which may occur by inactivation of nimTcdc25. Cells lacking both nimQMCM2 and bimEAPC1 are deficient in the S phase arrest checkpoint over mitosis because they lack both tyrosine phosphorylation of p34(cdc2) and the function of bimEAPC1. Initiation of DNA replication, which requires nimQMCM2, is apparently critical to switch mitotic regulation from the APC to include tyrosine phosphorylation of p34(cdc2) at G1/S. We also show that cells arrested at G1/S due to lack of nimQMCM2 continue to replicate spindle pole bodies in the absence of DNA replication and can undergo anaphase in the absence of APC function.