Activity of Novispirin G-10, a novel antimicrobial peptide against Chlamydia trachomatis and vaginosis-associated bacteria.

We tested the activity of Novispirin G-10, a novel antimicrobial alpha-helical octadecapeptide structurally related to cathelicidins and other innate immunity peptides, against Chlamydia trachomatis serovars L2, D, and E and three organisms associated with bacterial vaginosis (BV). The peptide's activity against C. trachomatis was measured in 48-h shell vial assays with McCoy cell targets. Exposure to 100 micro g/ml of Novispirin G-10 reduced the infectivity of serovars D and E by 99.4-100% and serovar L2 by 91.7-99.1%. At the same concentration of 100 micro g/ml, Novispirin G-10 rapidly killed >99% of Mobiluncus curtisii, Gardnerella vaginalis, and Prevotella bivia, in standard colony-forming unit (CFU) assays. Given its simple structure and relative lack of cytotoxic and hemolytic activity, Novispirin G-10 may be a useful component of microbicide preparations designed to prevent chlamydial infection and/or remediate the abnormal vaginal flora associated with BV.

The pig-tailed macaque rectal model: microflora and chlamydial infection.

BACKGROUND: A topical microbicide should protect against acquisition of sexually transmitted infection during both vaginal and rectal intercourse. The rectal microflora of the Macaca nemestrina (pig-tailed macaque) and humans were examined, as well as the histopathology of rectal tissues. In a subset of macaques, a human rectal isolate of Chlamydia trachomatis was inoculated into the rectum to establish rectal chlamydial infection. GOAL: To evaluate the comparability of the pig-tailed macaque rectal model with humans. STUDY DESIGN: Rectal swabs were collected for microbiologic analysis to characterize normal microflora in pig-tailed macaques and humans. Subsequently, 10 macaques received a rectal inoculation with C trachomatis, serovar D, prepared from a clinical rectal isolate. RESULTS: The rectal microflora of pig-tailed macaques (n = 80) were found to be comparable with the rectal flora of humans (n = 40). The prevalence of Lactobacillus in the rectum was higher in the macaques than in humans. Coliform and Enterococcus were decreased in the macaques, as compared with those of humans. In 9 of 10 macaques, rectal chlamydial infection was confirmed by culture or ligase chain reaction on days 2, 7, and 14 after inoculation. The test results were positive for rectal chlamydial infection by ligase chain reaction only for the remaining animal on day 14 after inoculation. CONCLUSIONS: The findings demonstrate that the rectal environment of the pig-tailed macaque is a useful model for further evaluation of newly developed topical microbicides for rectal use. Furthermore, such products can be evaluated for protection against rectal chlamydial infection in this model.

Multiple drug-resistant Chlamydia trachomatis associated with clinical treatment failure.

In vitro susceptibility testing and genotyping were done on urogenital isolates of Chlamydia trachomatis from 3 patients, 2 of whom showed evidence of clinical treatment failure with azithromycin and one of whom was the wife of a patient. All 3 isolates demonstrated multidrug resistance to doxycycline, azithromycin, and ofloxacin at concentrations >4.0 microg/mL. Recurrent disease due to relapsing infection with the same resistant isolate was documented on the basis of identical genotypes of both organisms. This first report of clinically significant multidrug-resistant C. trachomatis causing relapsing or persistent infection may portend an emerging problem to clinicians and public health officials.

Structural analysis of the lipopolysaccharide from Chlamydia trachomatis serotype L2.

The lipopolysaccharide (LPS) of Chlamydia trachomatis L2 was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. From a total of 5 x 10(4) cm2 of infected monolayers, 22.3 mg of LPS were obtained. Compositional analysis indicated the presence of 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), GlcN, phosphorus, and fatty acids in a molar ratio of 2.8:2:2.1:4.5. Matrix-assisted laser-desorption ionization mass spectrometry performed on the de-O-acylated LPS gave a major molecular ion peak at m/z 1781.1 corresponding to a molecule of 3 Kdo, 2 GlcN, 2 phosphates, and two 3-hydroxyeicosanoic acid residues. The structure of deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide was determined by 600 MHz NMR spectroscopy as Kdoalpha2-->8Kdoalpha2-->4Kdoalpha2-->6D-GlcpNbeta1 -->6D-GlcpNalpha 1,4'-bisphosphate. These data, together with those published recently on the acylation pattern of chlamydial lipid A (Qureshi, N., Kaltashov, I., Walker, K., Doroshenko, V., Cotter, R. J., Takayama, K, Sievert, T. R., Rice, P. A., Lin, J.-S. L., and Golenbock, D. T. (1997) J. Biol. Chem. 272, 10594-10600) allow us to present for the first time the complete structure of a major molecular species of a chlamydial LPS.