PREVALENCE OF CHLAMYDIACEAE AND TETRACYCLINE RESISTANCE GENES IN WILD BOARS OF CENTRAL EUROPE.

Our aim was to investigate the occurrence and distribution of Chlamydia suis and other Chlamydiaceae in the wild boar (Sus scrofa) population of Switzerland and Northern Italy and the detection of tetracycline resistance genes by PCR. We collected a total of 471 conjunctival swabs (n=292), rectal swabs (n=147), and lung tissue samples (n=32) belonging to 292 wild boars. The prevalence of Chlamydiaceae in the investigated wild boar populations was very low (1.4%, 4/292). We found C. suis in rectal or conjunctival swabs but not in lung samples. The low chlamydial prevalence might be attributed to limited contacts between wild boars and outdoor domestic pigs due to strict biosecurity measures or limited numbers of rural pig herds. The tetA(C) gene fragment was detected in six samples, which were all negative for Chlamydiaceae, and was probably not of chlamydial origin but more likely from other bacteria. The low tetracycline resistance rate in wild boar might be explained by the lack of selective pressure. However, transmission of resistance genes from domestic pigs to wild boar or selective pressure in the environment could lead to the development and spread of tetracycline-resistant C. suis strains in wild boars.

Azithromycin and Doxycycline Attenuation of Acanthamoeba Virulence in a Human Corneal Tissue Model.

Background: Amoebic keratitis is a potentially blinding eye infection caused by ubiquitous, free-living, environmental acanthamoebae, which are known to harbor bacterial endosymbionts. A Chlamydia-like endosymbiont has previously enhanced Acanthamoeba virulence in vitro. We investigated the potential effect of Acanthamoeba-endosymbiont coinfection in a human corneal tissue model representing clinical amoebic keratitis infection. Methods: Environmental and corneal Acanthamoeba isolates from the American Type Culture Collection were screened for endosymbionts by amplifying and sequencing bacterial 16S as well as Chlamydiales-specific DNA. Each Acanthamoeba isolate was used to infect EpiCorneal cells, a 3-dimensional human corneal tissue model. EpiCorneal cells were then treated with azithromycin, doxycycline, or control medium to determine whether antibiotics targeting common classes of bacterial endosymbionts attenuated Acanthamoeba virulence, as indicated by decreased observed cytopathic effect and inflammatory biomarker production. Results: A novel endosymbiont closely related to Mycobacterium spp. was identified in Acanthamoeba polyphaga 50495. Infection of EpiCorneal cells with Acanthamoeba castellanii 50493 and A. polyphaga 50372 led to increased production of inflammatory cytokines and cytopathic effects visible under microscopy. These increases were attenuated by azithromycin and doxycycline. Conclusions: Our findings suggest that azithromycin and doxycycline may be effective adjuvants to standard antiacanthamoebal chemotherapy by potentially abrogating virulence-enhancing properties of bacterial endosymbionts.

Chlamydial biology and its associated virulence blockers.

Chlamydiales are obligate intracellular parasites of eukaryotic cells. They can be distinguished from other Gram-negative bacteria through their characteristic developmental cycle, in addition to special biochemical and physical adaptations to subvert the eukaryotic host cell. The host spectrum includes humans and other mammals, fish, birds, reptiles, insects and even amoeba, causing a plethora of diseases. The first part of this review focuses on the specific chlamydial infection biology and metabolism. As resistance to classical antibiotics is emerging among Chlamydiae as well, the second part elaborates on specific compounds and tools to block chlamydial virulence traits, such as adhesion and internalization, Type III secretion and modulation of gene expression.

Antimicrobial susceptibility and molecular typing of multiple Chlamydiaceae species isolated from genital infection of women in Egypt.

This study investigated the existence of vaginal Chlamydia infection and the prevalence of the disease in symptomatic gynecologically diseased women in Egypt. In addition, the antibiotics penicillin, tetracycline, and erythromycin were evaluated for their in vitro antichlamydial activity of the isolated strains. Vaginal swabs (n=160) were collected from females gynecologically diseased using cotton swabs. Samples were tested for Chlamydia by Vero cells tissue culture, chicken embryo, Gimenez staining, direct fluorescein-conjugated monoclonal antibody staining, and immunoperoxidase. Polymerase chain reaction (PCR) analyses conducted for the presence of chlamydial DNA was used to detect its specific DNA by the omp2 gene. PCR analyses conducted for the presence of chlamydial DNA revealed that 112/160 (70%) were positive for Chlamydiaceae. The specific DNA defined by the omp2 gene identified them as Chlamydia trachomatis (17/112, 15.2%), Chlamydophila psittaci (56/112, 50.0%), and Chlamydophila abortus (40/112, 35.7%). The antibiotics penicillin, tetracycline, and erythromycin at different concentrations were effective in inactivating the viability of Chlamydiaceae isolates.

Reversal of the antichlamydial activity of putative type III secretion inhibitors by iron.

INPs, which are chemically synthesized compounds belonging to a class of acylated hydrazones of salicylaldehydes, can inhibit the growth of Chlamydiaceae. Evidence has been presented that in Yersinia and Chlamydia INPs may affect the type III secretion (T3S) system. In the present study 25 INPs were screened for antichlamydial activity at a concentration of 50 muM, and 14 were able to completely inhibit the growth of Chlamydia trachomatis serovar D in McCoy and HeLa 229 cells. The antichlamydial activities of two of these INPs, INPs 0341 and 0400, were further characterized due to their low cytotoxicity. These compounds were found to inhibit C. trachomatis in a dose-dependent manner; were not toxic to elementary bodies; were cidal at a concentration of > or =20 microM; inhibited all Chlamydiaceae tested; and could inhibit the development of C. trachomatis as determined by the yield of progeny when they were added up to 24 h postinfection. INP 0341 was able to affect the expression of several T3S genes. Compared to the expression in control cultures, lcrH-1, copB, and incA, all middle- to late-expressed T3S genes, were not expressed in the INP 0341-treated cultures 24 to 36 h postinfection. Iron, supplied as ferrous sulfate, as ferric chloride, or as holo-transferrin, was able to negate the antichlamydial properties of the INPs. In contrast, apo-transferrin and other divalent metal ions tested were not able to reverse the inhibitory effect of the INPs. In conclusion, the potent antichlamydial activity of INPs is directly or indirectly linked with iron, and this inhibition of Chlamydia has an effect on the T3S system of this intracellular pathogen.

Inclusion counter: a public domain image analysis system for measurement of Chlamydiaceae growth in vitro.

The traditional method of measuring chlamydial growth in vitro, counting Chlamydiaceae inclusions by eye, is time-consuming and error prone. This paper describes a novel automated image analysis system suitable for high-throughput screening of novel anti-Chlamydiaceae compounds. The software, Inclusion Counter v3.0, is freely available in the public domain (http://www.image-analysis.co.uk).

Optimal development of Chlamydophila psittaci in L929 fibroblast and BGM epithelial cells requires the participation of microfilaments and microtubule-motor proteins.

The cytoskeleton is involved in several cellular activities, including internalization and transport of foreign particles. Although particular functions to each cytoskeleton component have been described, interactions between those components seem to occur. The involvement of the different host cell cytoskeletal components in uptake and development of Chlamydophila psittaci is incompletely understood. In this study, the participation of the microfilament network along with the kinesin and dynein microtubule motor proteins in the internalization and further development of Chlamydophila psittaci were investigated in L929 fibroblast and BGM epithelial cells. Cytochalasin D disruption of actin filaments, and blockage of the motor proteins through the introduction of monoclonal antibodies into the host cells were carried out, either single or combined, at different moments around bacterial inoculation, and Chlamydophila infectivity determined 24 h post- inoculation by direct immunofluorescence. Our results show that, although Chlamydophila Ipsittaci can make use of both microfilament-dependent and independent entry pathways in both cell types, Chlamydophila internalization and development in the fibroblast cells mainly concerned processes mediated by microfilaments while in the epithelial cells mechanisms that require microtubule motor proteins were the ones predominantly involved. Evidence that mutual participation of the actin and tubulin networks in both host cells are required for optimal growth of Chlamydophila psittaci is also presented.