David W. Hilbert

Uropathogenic Escherichia coli dominantly suppress the innate immune response of bladder epithelial cells by a lipopolysaccharide- and Toll-like receptor 4-independent pathway.

Urinary tract infections are a major source of morbidity among women, with the majority caused by uropathogenic Escherichia coli. Our objective was to test if uropathogenic E. coli suppress the innate immune response of bladder epithelial cells. We found that bladder epithelial cells secrete interleukin-6 and interleukin-8 in response to non-pathogenic E. coli, whereas they failed to do so in response to uropathogenic E. coli. Uropathogenic E. coli prevented interleukin-6 secretion in response to non-pathogenic E. coli and a panel of Toll-like receptor agonists, as well as to interleukin-1beta, but not to tumor necrosis factor alpha. These results indicate that receptors with a Toll/interleukin-1 receptor domain are specifically targeted, and that suppression is not a consequence of toxicity. One candidate for mediating immune suppression is bacterial lipopolysaccharide. However, lipopolysaccharide isolated from either uropathogenic or non-pathogenic E. coli stimulated interleukin-6 secretion to similar levels. In addition, uropathogenic E. coli did not stimulate interleukin-6 secretion from cells expressing a dominant negative Toll-like receptor 4, and prevented cells lacking Toll-like receptor 4 from secreting interleukin-6 in response to synthetic lipoprotein. We conclude that uropathogenic E. coli suppress the innate immune response through a pathway partially independent of lipopolysaccharide and Toll-like receptor 4.

Detecting DNA methylation of the BCL2, CDKN2A and NID2 genes in urine using a nested methylation specific polymerase chain reaction assay to predict bladder cancer.

PURPOSE Detection of methylated DNA has been shown to be a good biomarker for bladder cancer. Bladder cancer has the highest recurrence rate of any cancer and, as such, patients are regularly monitored using invasive diagnostic techniques. As urine is easily attainable, bladder cancer is an optimal cancer to detect using DNA methylation. DNA methylation is highly specific in cancer detection. However, it is difficult to detect because of the limited amount of DNA present in the urine of patients with bladder cancer. Therefore, an improved, sensitive and noninvasive diagnostic test is needed. MATERIALS AND METHODS We developed a highly specific and sensitive nested methylation specific polymerase chain reaction assay to detect the presence of bladder cancer in small volumes of patient urine. The genes assayed for DNA methylation are BCL2, CDKN2A and NID2. The regions surrounding the DNA methylation sites were amplified in a methylation independent first round polymerase chain reaction and the amplification product from the first polymerase chain reaction was used in a real-time methylation specific polymerase chain reaction. Urine samples were collected from patients receiving treatment at Wolfson Medical Center in Holon, Israel. RESULTS In a pilot clinical study using patient urine samples we were able to differentiate bladder cancer from other urogenital malignancies and nonmalignant conditions with a sensitivity of 80.9% and a specificity of 86.4%. CONCLUSIONS We developed a novel methylation specific polymerase chain reaction assay for the detection and monitoring of bladder cancer using DNA extracted from patient urine. The assay may also be combined with other diagnostic tests to improve accuracy.

Trichomonas vaginalis Metronidazole Resistance Is Associated with Single Nucleotide Polymorphisms in the Nitroreductase Genes ntr4Tv and ntr6Tv

ABSTRACT Metronidazole resistance in the sexually transmitted parasite Trichomonas vaginalis is a problematic public health issue. We have identified single nucleotide polymorphisms (SNPs) in two nitroreductase genes (ntr4Tv and ntr6Tv) associated with resistance. These SNPs were associated with one of two distinct T. vaginalis populations identified by multilocus sequence typing, yet one SNP (ntr6Tv A238T), which results in a premature stop codon, was associated with resistance independent of population structure and may be of diagnostic value.

Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis.

ABSTRACT Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsels criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease: Gardnerella vaginalis, Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the order Clostridiales), Megasphaera phylotype 1 or 2, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. We generated a logistic regression model that identified G. vaginalis, A. vaginae, and Megasphaera phylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsels criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion of Lactobacillus spp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV.

Identification of intrinsically metronidazole-resistant clades of Gardnerella vaginalis.

Gardnerella vaginalis is associated with bacterial vaginosis (BV), the most common cause of vaginal discharge. Metronidazole is a front-line therapy for BV, and treatment failure and recurrent disease are common problems. Whole-genome sequencing studies have revealed that G. vaginalis has a population structure that consists of 4 clades: clades 1 and 3 are associated with BV, whereas clades 2 and 4 are not. To determine if metronidazole susceptibility is associated with population structure, we analyzed 87 clinical isolates and found that metronidazole resistance (MIC ≥32 μg/mL) was highly associated with clade (P<0.0001), as 14/14 clade 3 isolates (100%) and 22/22 clade 4 isolates (100%) exhibited resistance, compared to only 16/37 clade 1 isolates (35%) and 1/14 clade 2 isolates (7.1%). The identification of intrinsically metronidazole-resistant G. vaginalis clades will facilitate future studies on the relationship between metronidazole resistance and BV treatment failure.

Cervical and Vaginal Flora Specimens Are Highly Concordant with Respect to Bacterial Vaginosis-Associated Organisms and Commensal Lactobacillus Species in Women of Reproductive Age

ABSTRACT Matched vaginal and cervical specimens from 96 subjects were analyzed by quantitative PCR for the presence and concentration of bacterial vaginosis-associated microbes and commensal Lactobacillus spp. Detection of these microbes was 92% concordant, indicating that microbial floras at these body sites are generally similar.

Utilization of molecular methods to identify prognostic markers for recurrent bacterial vaginosis

BACKGROUND Recurrent bacterial vaginosis (BV) after antimicrobial therapy is a major problem, affecting >50% of patients within 1 year. The objective of this study was to determine if prospective identification of patients at risk for recurrence using molecular methods is feasible. METHODS Women were evaluated for BV by Amsel criteria and Nugent score. Vaginal specimens were analyzed using a panel of quantitative real-time polymerase chain reactions (qPCRs) at three times: pre-treatment, 7-10days post-treatment and 40-45days post-treatment. The PCRs quantified DNA of the following organisms: Gardnerella vaginalis; Atopobium vaginae; Bacterial Vaginosis-Associated Bacteria-1 (BVAB1), -2 (BVAB2) and -3 (BVAB3); Leptotrichia/Sneathia; Megasphaera Phylotypes 1 and 2; and Lactobacillus spp. (L. crispatus, L. gasseri, L. iners and L. jensenii). RESULTS Out of 84 women diagnosed with BV (Amsel ≥3 and Nugent ≥4), 77 (91.7%) were successfully treated after 7-10days (asymptomatic and Amsel of either 0 or 1 with elevated vaginal pH and Nugent ≤6). Of these 77 women, 46 (59.7%) remained cured after 40-45days and 31 (40.3%) developed recurrent BV. In univariate analysis, we found that women who would have recurrent BV during the study had greater concentrations of Megasphaera Phylotype 2 (P=0.001) and BVAB2 (P=0.015) at initial diagnosis and greater vaginal pH (P=0.030), higher Nugent score (P=0.043) and a greater concentration of G. vaginalis (P=0.012) post-treatment, when compared to women who were cured during the study. These differences largely remained when cure was defined as Nugent ≤3 or when only women treated with intravaginal metronidazole were evaluated. CONCLUSION Molecular analysis of BV is a useful adjunct to clinical and microscopic analysis to prospectively identify patients at high risk for recurrent BV.

Draft Genome Sequence of a Metronidazole-Resistant Derivative of Gardnerella vaginalis Strain ATCC 14019

ABSTRACT We report the genome sequence of a metronidazole-resistant derivative of Gardnerella vaginalis ATCC 14019. This strain was obtained after serial selection to increase the MIC from 4 to ≥500 µg/ml. Two coding changes, in genes encoding a response regulator and an NAD+ synthetase, arose during selection.

Draft Genome Sequence of a Metronidazole-Susceptible Atopobium vaginae Isolate

ABSTRACT We report the draft genome sequence of a vaginal isolate of Atopobium vaginae vaginae (strain 44061), an organism linked to bacterial vaginosis (BV), the most common gynecological infection in the United States. This species is often highly resistant to metronidazole, which is a front-line therapy for BV. Strain 44061 is a metronidazole-susceptible isolate (MIC, 16 µg/ml), and its genome sequence will be useful for comparative studies to elucidate the molecular basis of metronidazole resistance in this species.

Draft Genome Sequence of a Metronidazole-Resistant Gardnerella vaginalis Isolate.

ABSTRACT We report the draft genome sequence of a Gardnerella vaginalis strain (3549624) isolated from a vaginal specimen. G. vaginalis is associated with bacterial vaginosis, the most common cause of vaginal discharge, which is often treated with metronidazole. This isolate is highly resistant to metronidazole (MIC, 500 µg/ml) and may be useful for comparative genomic studies to determine the molecular basis of metronidazole resistance in this species.